CA3199484A1 - Biomarkers for predicting immunogenicity and therapeutic responses to adalimumab in rheumatoid arthritis patients - Google Patents

Biomarkers for predicting immunogenicity and therapeutic responses to adalimumab in rheumatoid arthritis patients

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CA3199484A1
CA3199484A1 CA3199484A CA3199484A CA3199484A1 CA 3199484 A1 CA3199484 A1 CA 3199484A1 CA 3199484 A CA3199484 A CA 3199484A CA 3199484 A CA3199484 A CA 3199484A CA 3199484 A1 CA3199484 A1 CA 3199484A1
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antigens
adalimumab
response
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biomarkers
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Jonathan Michael Blackburn
Arif Anwar
Der-Yuan Chen
Po-Ku CHEN
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Sengenics Corp Pte Ltd
China Medical University Hospital
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

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Abstract

A method for predicting an immunogenic and/or therapeutic response to adalimumab 5 from a sample extracted from a rheumatoid arthritis patient by testing the sample for the presence of biomarkers, the biomarkers being autoantibodies to antigens comprising SSB, TROVE2 and ZHX2.

Description

2 BIOMARKERS FOR PREDICTING IMMUNOGENICITY AND
THERAPEUTIC RESPONSES TO ADALIMUMAB IN RHEUMATOID
ARTHRITIS PATIENTS
Field of Invention The invention relates to the detection of immunological biomarkers, particularly autoantibodies, to predict immunogenicity and therapeutic responses to adalimumab in patients with Rheumatoid Arthritis (RA).
Background Rheumatoid arthritis (RA), a chronic inflammatory articular disease, is characterized by persistent synovitis, cartilage degradation, and bone erosions [1], and tumor necrosis factor (TNF)-a is a crucial inflammatory mediator in RA-related synovitis and joint damage [2]. The importance of the role of TNF-a in RA pathogenesis is supported by the effectiveness of biologics targeting this cytokine [2-4], although the efficacy diminishes in some patients over time (secondary failure) [5]. Accumulating evidence indicates that the presence of anti-drug antibodies (ADAb) in certain patients may be associated with low or undetectable drug levels and ensuing reduction of therapeutic responsiveness to TNF-ot inhibitors [6-10] Such ADAb responses reflect the differential immunogenicity of the given biologic drug triggered in individual patients, which results in some patients developing a neutralising antibody response against the biologic drug and others not. In the face of such uncertainty about whether individual RA patients will show therapeutic responsiveness to TNF-a inhibitors or not [11], physicians hoping to optimize personalized and precision therapy are thus eager to find biomarkers which can predict the emergence of ADAb and the effectiveness of anti-TNF-a biologics.
Proteomics research has been increasingly applied to the identification of novel biomarkers that might be useful for monitoring therapeutic response in RA
patients on specific treatments [12-14] However, there is currently limited knowledge about circulating biomarkers that are able to predict the development of ADAb in RA
patients receiving anti-TNF-cc therapy.

Autoantibody biomarkers as described herein are autoantibodies to antigens, autoantibodies being antibodies which are produced by an individual which are directed against one or more of the individual's own proteins (self' antigens).
The aim of the present invention therefore is to provide a novel panel of autoantibody biomarkers that are able to predict immunogenicity of adalimumab and therapeutic responses to adalimumab in individual RA patients, prior to treatment with adalimumab, a widely used TNF-a inhibitor marketed under the brand name HUMIRA and commonly used for the treatment of autoimmune diseases, such as RA, Crohn's Disease 1 0 and Psoriasis.
Summary of Invention In one aspect of the invention, there is provided a method for predicting a response to adalimumab from a sample extracted from a rheumatoid arthritis patient prior to treating the patient with adalimumab, said response being classified as a good response corresponding to anti-drug antibody negative or a poor response corresponding to anti-drug antibody positive, comprising the steps of:
(i) testing the sample for the presence of autoantibody biomarkers; and (ii) determining whether the patient will develop a good response or a bad response to treatment with adalimumab, based on the detection of said autoantibody biomarkers;
characterised in that the autoantibody biomarkers are autoantibodies to antigens comprising SSB, TROVE2 and ZHX2, wherein ZHX2 is associated with the good response, and SSB and TROVE2 are associated with the poor response.
Advantageously the autoantibody biomarkers can be used to predict immunogenicity of adalimumab and therapeutic responses to adalimumab in individual rheumatoid arthritis (RA) patients at baseline (i.e. prior to treating the patient with adalimumab) In one embodiment the sample is tested using a panel of antigens that correspond to the autoantibody biomarkers. Typically the antigens are biotinylated proteins.
Advantageously the bi otinyl ati on ensures that the antigens are folded in their correct form to ensure accuracy of detection by the autoantibody biomarkers.
3 In one embodiment the antigens may include one or more additional antigens from the group comprising of PPARD, SPANXN2, HNRNPA2B, TRIB2, CEP55, SH3GL1, FN3K, PANK3, HPCAL1, THRA, AIFM1, ODC1, RPS6KA4, EEF1D, KLF10, EPHA2, PRKAR1A and EAPP
It should be noted that not all human antigens generate an autoantibody response and it is not possible to predict a priori which human antigens will do so in a given patient cohort ¨ of the 1622 antigens tested, only autoantibodies against the 21 antigens described above are suitable as biomarkers in predicting immunogenicity of adalimumab and therapeutic responses to adalimumab in RA patient at baseline.
In one embodiment each biotinylated protein is formed from a Biotin Carboxyl Carrier Protein (BCCP) folding marker which is fused in-frame with the protein.
In one embodiment the biotinylated proteins are bound to a streptavidin-coated substrate.
Advantageously full-length proteins are expressed as fusions to the BCCP
folding marker which itself becomes biotinylated in vivo when the fusion partner is correctly folded By comparison misfolded fusion partners cause the BCCP to remain in the `apo' (i.e. non-biotinylated) form such that it cannot attach to a streptavidin substrate.
Thus only correctly folded fusion proteins become attached to the streptavidin substrate via the biotin moiety appended to the BCCP tag.
In one embodiment the substrate comprises a glass slide, biochip, strip, slide, bead, microtitre plate well, surface plasmon resonance support, microfluidic device, thin film polymer base layer, hydrogel-forming polymer base layer, or any other device or technology suitable for detection of antibody-antigen binding In one embodiment the substrate is exposed to a sample extracted from a person, such that autoantibody biomarkers from the sample may bind to the antigens.
4 Typically the sample comprises any or any combination of exosomes, blood, serum, plasma, urine, saliva, amniotic fluid, cerebrospinal fluid, breast milk, semen or bile Typically the sample is collected at baseline prior to administration of the first dose of adalimumab.
In one embodiment following exposure to the sample, the substrate is exposed to a fluorescently-tagged secondary antibody to allow the amount of any autoantibodies from the sample bound to the antigens on the panel to be determined. Typically the secondary antibody is anti-human IgG, but it will be appreciated that other secondary antibodies could be used, such as anti-IgM, anti-IgGl, anti-IgG2, anti-IgG3, anti-IgG4 or anti-IgA.
In one embodiment the patient's response to treatment with adalimumab (i.e.
the immunogenic and/or therapeutic response outcome to adalimumab in RA patient at baseline) corresponds to the relative or absolute amount of autoantibodies from the baseline sample specifically binding to the antigens.
In one embodiment the method is performed in vitro.
In a further aspect of the invention, there is provided a method for manufacturing a kit for predicting a response to adalimumab from a sample extracted from a rheumatoid arthritis patient prior to treating the patient with adalimumab, comprising the steps of:
for each antigen in a panel, cloning a biotin carboxyl carrier protein folding marker in-frame with a gene encoding the antigen and expressing the resulting biotinylated antigen;
binding the biotinylated antigens to addressable locations on one or more streptavidin-coated substrates, thereby forming an antigen array, such that the amount of autoantibodies from the sample binding to the antigens on the panel can be determined by exposing the substrate to the sample and measuring the immunogenicity and response, characterised in that the antigens comprise SSB, TROVE2 and ZHX2.

In one embodiment the antigens further comprise one or more of PPARD, SPANXN2, HNRNPA2B, TRIB2, CEP55, SH3GL1, FN3K, PANK3, HPCAL1, TEMA, AIFM1, ODC1, RP S6KA4, EEF1D, KLF10, EPHA2, PRKAR1A and EAPP.
5 In a further aspect of the invention there is provided a method for predicting immunogenicity of adalimumab and therapeutic responses to adalimumab in RA
patients at baseline by exposing a composition comprising a panel of antigens as herein described to a sample extracted from a person, and determining the level of autoantibodies from the sample binding to the antigens.
In a yet further aspect of the invention there is provided a method for predicting immunogenicity of adalimumab and therapeutic responses to adalimumab in RA
patients at baseline by exposing a composition comprising a panel of antigens as herein described to a sample extracted from a person in vitro, and determining the level of autoantibodies from the sample binding to the antigens In further aspect of the invention, there is provided a composition comprising a panel of antigens for predicting an immunogenic and/or therapeutic response to adalimumab in a rheumatoid arthritis patient who has not previously been treated with adalimumab, characterised in that the antigens comprise SSB, TROVE2 and ZHX2.
In one embodiment the antigens further comprise one or more of PPARD, SPANXN2, HNRNPA2B, TRIB2, CEP55, SH3GL1, FN3K, PANK3, HPCALL THRA, AIFM1, ODC1, RP S6KA4, EEF1D, KLF10, EPHA2, PRKAR1A and EAPP.
In one embodiment the antigens are biotinylated proteins In one embodiment the amount of one or more autoantibody biomarkers binding in vitro to the antigens in a sample from a patient can be measured to determine the immunogenicity and therapeutic response outcome to adalimumab in an RA patient at baseline.
6 In yet further aspect of the invention, there is provided a composition comprising a panel of autoantibody biomarkers for predicting an immunogenic and/or therapeutic response to adalimumab in a rheumatoid arthritis patient who has not previously been treated with adalimumab, wherein the level of the autoantibody biomarkers are measured in a sample collected from the patient;
characterised in that the autoantibody biomarkers are specific to antigens comprising SSB, TROVE2 and ZHX2.
Brief Description of Drawings It will be convenient to further describe the present invention with respect to the accompanying drawings that illustrate possible arrangements of the invention.
Other arrangements of the invention are possible, and consequently the particularity of the accompanying drawings is not to be understood as superseding the generality of the preceding description of the invention.
Figure 1 illustrates the structure of the E. coli Biotin Carboxyl Carrier Protein domain.
Figure 2 illustrates the pPRO9 plasmid used as a vector.
Figure 3 illustrates the distribution of the normalised RFU (i.e. autoantibody responses) for all 21 biomarkers between ADAb-positive (Group A) and ADAb-negative (Group B) RA patients collected at baseline.
Figure 4 illustrates the discriminatory performance represented as a receiver operating curve (ROC) with an area under the curve (AUC) of 0.835 for all 21 biomarkers (SSB, TROVE2, ZHX2, PPARD, SPANXN2, HNRNPA2B, TRIB2, CEP55, SH3GL1, FN3K, PANK3, HPCAL1, THRA, ALFM1, ODC1, RP S6KA4, EEF1D, KLF10, EPHA2, PRKAR1A and EAPP) between ADAb-positive (Group A) and ADAb-negative (Group B) RA patients collected at baseline Figure 5 illustrates the variable importance measure for each of the 21 biomarkers identified in the study.
7 Figure 6 illustrates the discriminatory performance represented as a receiver operating curve (ROC) with an area under the curve (AUC) of 0.761 for the core biomarkers (SSB, TROVE2 and ZHX2) between ADAb-positive (Group A) and ADAb-negative (Group B) RA patients collected at baseline.
Detailed Description The invention utilises the Biotin Carboxyl Carrier Protein (BCCP) folding marker which is cloned in-frame with the gene encoding the protein of interest, as described above and in EP1470229. The structure of the E. coli BCCP domain is illustrated in Figure 1, wherein residues 77-156 are drawn (coordinate file lbdo) showing the N- and C-termini and the single biotin moiety that is attached to lysine-122 in vivo by biotin ligase.
BCCP acts not only as a protein folding marker but also as a protein solubility enhancer.
BCCP can be fused to either the N- or C-terminal of a protein of interest.
Full-length proteins are expressed as fusions to the BCCP folding marker which becomes biotinylated in vivo, but only when the protein is correctly folded. Conversely, misfolded proteins drive the misfolding of BCCP such that it is unable to become biotinylated by host biotin ligases. Hence, misfolded proteins are unable to specifically attach to a streptavidin-coated solid support. Therefore only correctly folded proteins become attached to a solid support via the BCCP tag.
The surface chemistry of the support is designed carefully and may use a three-dimensional thin film hydrogel layer (polyethylene glycol; PEG), which retains protein spot morphologies and ensures consistent spot sizes across the array. The PEG
layer inhibits non-specific macromolecule absorption, therefore reducing the high background observed using other platforms. The solid support used to immobilize the selected biomarkers thus provides excellent signal-to-noise ratios and low limits of detection (translating in to improved sensitivity). In addition the PEG hydrogel layer also aids preservation of the folded structure and functionality of arrayed proteins and protein complexes post-immobilisation.
Retention of the correct folded structure of immobilised antigens during antibody binding assays ('immuno-assays') is particularly advantageous because human antibodies are
8 known in general to specifically recognise and bind to discontinuous, solvent-accessible epitopes on protein surfaces, yet are also known to bind non-specifically to exposed hydrophobic surfaces on unfolded proteins. Thus serological assays carried out on arrays of unfolded proteins typically give rise to many false positive results due to such non-specific binding events (which have no biological relevance), whilst at the same time also giving rise to many false negative results due to the absence of biologically-relevant discontinuous epitopes. By contrast, serological assays carried out on arrays of folded antigens result in detection of biologically meaningful antibody-antigen interactions that are not obscured by high rates of non-specific binding.
As biotinylated proteins bound to a streptavidin-coated surface show negligible dissociation, this interaction therefore provides a superior means for tethering proteins to a planar surface in a controlled orientation and is thus ideal for applications such as protein arrays, SPR and bead-based assays. The use of a compact, folded, biotinylated, 80 residue domain BCCP affords two significant advantages over for example the AviTag and intein-based tag. First, the BCCP domain is cross-recognised by eukaryotic biotin ligases enabling it to be biotinylated efficiently in yeast, insect, and mammalian cells without the need to co-express the E. coli biotin ligase. Second, the N- and C-termini of BCCP are physically separated from the site of biotinylation by 50A (as shown in Figure 1), so the BCCP domain can be thought of as a stalk which presents the recombinant proteins away from the solid support surface, thus minimising any deleterious effects due to immobilisation.
The addition of BCCP permits the monitoring of fusion protein folding by measuring the extent of in vivo biotinylation. This can be measured by standard blotting procedures, using SDS-PAGE or in situ colony lysis and transfer of samples to a membrane, followed by detection of biotinylated proteins using a streptavidin conjugate such as streptavidin-horseradish peroxidase. Additionally, the fact that the BCCP domain is biotinylated in vivo is particularly useful when multiplexing protein purification for fabrication of protein arrays since the proteins can be simultaneously purified from cellular lysates and immobilised in a single step via the high affinity and specificity exhibited by a streptavi din surface.
9 Example 1 Materials and Methods Gene synthesis and cloning. The pPRO9 plasmid (see Figure 2 below) was constructed by standard techniques and consists of genetic elements encoding a c-myc tag and a BCCP protein domain, preceded by a multiple-cloning site. Synthetic genes encoding individual human antigens were assembled from synthetic oligonucleotides and were cloned into pPRO9 using Spel and Ncol cloning sites such that each resultant clonal 'transfer vector' encoded an in-frame fusion protein comprising a specific human antigen fused to the BCCP tag. The plasmid DNA was purified from transformed bacteria and verified by DNA sequencing. The required sequence congruence within the synthetic gene region was 100%.
Recombinant baculovirus was generated via co-transfection of Sf9 cells (a clonal isolate derived from the parental Spot/op/era frugiperda cell line IPLB-Sf-21-AE) with a replication-deficient bacmid vector carrying the viral polyhedrin promoter and a transfer vector carrying a specific coding sequence for a specific antigen. Homologous recombination between the transfer vector and the bacmid within Sf9 cells resulted in formation of a replication competent baculoviral vector encoding the specific antigen fused to the BCCP tag. Successful homologous recombination between the transfer vector and the bacmid within Sf9 cells caused the transfected cells to show signs of viral cytopathic effect (CPE) within few days of culture incubation. The most common CPE
observed was the significantly enlargement of average cell size, a consequences of viral progeny propagation. These baculoviruses known as PO were then released into the culture medium, and viral amplification were done to generate a higher titre of P1 viruses.
Protein Expression. Expression of recombinant antigens was carried out in 24 well blocks using 3m1 cultures containing 6x106 Sf9 cells per well. High titre, low passage, viral stocks of recombinant baculovirus (>107 pfu/ml) were used to infect SP) insect cells.
The infected cells were then cultured for 72 hours to allow them to produce the recombinant protein of interest. The cells were washed with PBS, resuspended in buffer, and were frozen in aliquots at -80 C ready for lysis as required. Depending on the transfer vector construct and the nature of the antigen itself, the resultant recombinant protein lysate can be recovered either from the cultured cell or the culture medium.
Expression of recombinant proteins was confirmed by SDS-PAGE as well as by Western blot using streptavidin-HRP-based detection. In total, 1622 human antigens were cloned and expressed using this methodology.

Array fabrication. Hydrogel coated, streptavidin-derivatised slides were custom manufactured by Schott and used as substrates on to which the biotinylated proteins were then printed. A total of 9 nanoliters of crude protein lysate was printed on a HS slide in quadruplicate using non-contact piezo printing technology. Print buffer that have a pH
10 between 7.0 and 7.5 were used. The slides were dried by centrifugation (200 x g for 5 min) before starting the washing and blocking. The printed arrays were blocked with solutions containing BSA or casein (concentration: 0.1 mg/ml) in a phosphate buffer. The pH was adjusted to be between 7.0 and 7.5 and cold solutions were used (4 C -20 C).
Slides were not allowed to dry between washes, and were protected from light.
In total, each resultant 'Immunome array' comprised 1622 antigens, each printed in quadruplicate.
Experimental Procedure.
1. Study cohort The study cohort comprised of a total of 62 plasma and serum samples collected from RA
patients at baseline (i.e. prior to treatment administration);
i. Run 1. 6 ADAb-positive ("poor response") and 6 ADAb-negative ("good response") ii Run 2. 24 ADAb-positive ("poor response") and 26 ADAb-negative ("good response") Patients were administered with adalirnumab at a dose of 40mg every other week. The immunogenicity and therapeutic response to adalimumab were evaluated at week 24, the latter by using the EULAR response criteria [15]. EULAR responders were defined as RA patients with good and moderate ("good response") or poor ("poor response") EULAR therapeutic responses.
2. Sample Collection and Storage
11 Peripheral blood samples were collected immediately before the first adalimumab administration (the baseline sample) and also at week 24. After centrifugation at 1000 g for 10 min within 15 min of withdrawal, serum and plasma samples were stored at -70 C.
3. Sample Preparation and Dilution For each run, samples were placed in a shaking incubator set at 20 C to allow thawing for 30 minutes. When completely thawed, each sample was vortexed vigorously three times and debris was pelleted by centrifugation for 3 minutes at 13,000 rpm.
11.25 [IL of the sample was pipetted into 4.5 mL of Serum Assay Buffer (SAB) containing 0.1% v/v Triton, 0.1% w/v BSA in PBS (20 C) and vortexed to mix three times. The tube was tilted during aspiration to ensure that the sera was sampled from below the lipid layer at the top but did not touch the bottom of the tube in case of presence of any sediment.
Batch records were marked accordingly to ensure that the correct samples were added to the correct tubes. Samples were then randomised prior to assay.
4. Biomarker Assay Each Immunome array was removed from the storage buffer using forceps, placed in the slide box and rack containing 200 mL cold SAB and shaken on an orbital shaker at 50 rpm, for 5 minutes. After washing, each slide was scanned using a barcode scanner and then placed array side up in an individual slide hybridization chamber containing an individual diluted sera (Step 3 above). All slides were and incubated on a horizontal shaker at 50 rpm for 2 hours at 20 C.
5. Array Washing After Serum Binding Each Immunome array slide was rinsed twice in individual "Pap jars- with 30 mL
SAB, followed by 200 mL of SAB buffer in the slide staining box for 20 minutes on the shaker at 50 rpm at room temperature. All slides were transferred sequentially and in the same orientation.
6. Incubation with Cy3-anti-human IgG
Binding of autoantibodies to the arrayed antigens on the arrays was detected by incubation with Cy3-rabbit anti-human IgG (Dako Cytomati on) labelled according to the manufacturer's recommended protocols (GE Healthcare). Arrays were immersed in
12 hybridization solution containing a mixture of Cy3- rabbit anti-human IgG
solution diluted 1:1000 in SAB buffer for 2 hours at 50 rpm in 20 C.
7. Washing After Incubation with Cy3-anti-human IgG
After incubation, the slide was dipped in 200 mL of SAB buffer, 3 times for 5 minutes at 50 rpm at room temperature. Excess buffer was removed by immersing the slide in 200 mL of pure water for a few minutes. Slides were then dried for 2 min by centrifugation at 240g at room temperature. Slides were then stored at room temperature until scanning.
Fluorescent hybridization signals were measured with excitation at 550nm and emission at 570nm using a microarray laser scanner (Agilent) at 10p,m resolution.
Bioinformatic analysis.
1. Image Analysis: Raw Data Extraction The aim of an image analysis is to evaluate the amount of autoantibody present in the serum sample by measuring the median intensities of all the pixels within each probed spot. A raw .tiff format image file is generated for each slide, i.e. each sample. Automatic extraction and quantification of each spot on the array are performed using the GenePix Pro 7 software (Molecular Devices) which outputs the statistics for each probed spot on the array. This includes the mean and median of the pixel intensities within a spot as well as in its surrounding local background area. A GAL (GenePix Array List) file for the array is generated to enable image analysis. This file contains the information of all probed spots and their positions on the array. Following data extraction, a GenePix Results (.GPR) file is generated for each slide which contains the information for each spot: Protein ID, protein name, foreground intensities, background intensities etc. In the data sheet generated from the experiment, both foreground and background intensities of each spot are represented in relative fluorescence units (RFUs).
2. Data Handling and Pre-processing For each slide, antigens and control probes are spotted in quadruplicate on each array.
The following steps were performed to verify the quality of the antigen array data before proceeding with data analysis:
13 Step 1:
Calculate net intensities for each spot by subtracting background signal intensities from the foreground signal intensities of each spot. For each spot, the background signal intensity was calculated using a circular region with three times the diameter of the spot, centered on the spot.
Step 2:
Remove replica spots with net intensity < 0.
Step 3:
Zero net intensities if only 1 replica spot remaining.
Step 4:
Calculate the coefficient of variant (CV%) for the replica spots on each array.
S. D.
CV% = x 100%
Equation 1 Mean Flag any replica spots with only 2 or less replica/s remaining and CV% > 20%
as "High CV". The mean net intensity of such replica spots (i.e. antigens) is excluded from downstream analysis.
For antigens/controls with a CV% > 20% and with 3 or more replica spots remaining, the replica spots which result in this high CV% value were filtered out. This was done by calculating the standard deviation between the median value of the net intensities and individual net intensities for each set of replica spots. The spot with the highest standard deviation was removed. CV% values were re-calculated and the process repeated.
Step 5:
Calculate the mean of the net intensities for the remaining replica spots.
Step 6:
Inspect signal intensities of two positive controls: IgG and Cy3-BSA.
14 Step 7:
Carry out a composite normalisation [16] using both quantile-based and total intensity-based modules for each dataset . This method assumes that different samples share a common underlying distribution of their control probes while taking into account the potential existence of flagged spots within them. The Immunome array uses Cy3-labelled biotinylated BSA (Cy3-BSA) replicates as the positive control spots across slides. Hence it is considered as a 'housekeeping' probe for normalisation of signal intensities for any given study.
The quantile module adopts the algorithm described by Bolstad et al., 2003 [17]. This reorganisation enables the detection and handling of outliers or flagged spots in any of the Cy3BSA control probes. A total intensity-based module was then implemented to obtain a scaling factor for each sample. This method assumes that post-normalisation, the positive controls should have a common total intensity value across all samples. This composite method aims to normalise the protein array data from variations in their measurements whilst preserving the targeted biological activity across samples. The steps are as follows:
Quantile-Based Normalisation of all cy3BSA across all samples (i = spot number and] = sample number) 1. Load all Cy3-BSA across all samples, j, into an i X j matrix X
2. Sort spot intensities in each column j of X to get Xsoa 3. Take the mean across each row i of Xsort to get < Xe >
Intensity-Based Normalisation 1. Calculate sum of the mean across each row i , < Xi >
2. For each sample, k, calculate the sum of all Cy3-BSA controls, EXk 3. For each sample, k, E < xi >
Equation 2 Scaling factor (k) =
E Xk 3. Data Analysis Batch normalisation: The composite normalised data sets from the assays in the two runs were merged using a ComBat normalisation method [18]. For each protein, this method inputs the net intensity values across all the samples from the 2 data sets and adjusts for any possible batch effects between the two data sets using a parametric 5 empirical B ay e s frameworks.
Biomarker Panel Selection: A pipeline was developed which utilises a combination of feature selection and machine learning methodologies to determine the optimal combination of antigens eliciting autoantibody responses from the list of 1622 antigens 10 which are able to provide the best stratification between ADAb-positive and ADAb-negative patients [19]. For feature selection, univariate statistical tests, random forest importance and mutual information metrics were the filter methods used to rank biomarkers.
15 Biomarker panels were generated by additively selecting the top-ranking biomarkers as inputs to machine learning models up to a total of top 160 biomarkers (top 10%
of biomarkers). Any further addition of number of biomarkers did not lead to significant improvements of model performance and would lead to further increase of computational time. To estimate the biomarker panel performance, ROC, sensitivity and specificity was evaluated and the biomarker panel with the best sensitivity and specificity was deemed as the optimal panel to stratify ADAb status. For this analysis, machine learning models were built using Random Forests [20], under default settings with leave-one out cross validation (LOOCV). All analyses were performed using packages available in R.
Feature selection was performed using ranger [21] package and all machine learning models were performed using the caret [22] package.
Table 1 shows top 4 best performing biomarker panel from the machine learning models using leave-one out cross validation. The lowest number of antigens with the highest sensitivity and specificity was deemed to be the top biomarker panel. This panel comprises SSB, TROVE2, ZHX2, PPARD, SPANXN2, HNRNPA2B, TRI132, CEP55, SH3GL1, FN3K, PANK3, HP CALI, THRA, AIFM1, ODC1, RP S6KA4, EEF 1D, KLF10, EPHA2, PRK ARIA and EAPP. Figure 3 shows the distribution of the normalised net intensity (i.e. autoantibody responses) for each of these 21 individual biomarkers in
16 ADAb-positive (Group A) and ADAb-negative (Group B) RA patients at baseline.
Figure 4 shows the discriminatory performance of the combined panel of 21 autoantibody biomarkers represented as a receiver operating curve (ROC), yielding an area under the curve (AUC) of 0.835.
The biomarkers were ranked based on Random Forests estimated variable importance measure [23] derived from each panel (Figure 5 and Table 2). This further identified a core set of biomarkers which are common across the top 4 biomarker panels, comprising SSB, TROVE2 and ZHX2, with an AUC performance of 0.761 (Figure 6).
References 1. Choy EH, Panayi GS. Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med 2001;344:907-916.
2. Furst DE, Emery P. Rheumatoid arthritis pathophysiology: update on emerging cytokine and cytokine-associated cell targets. Rheumatology (Oxford) 2014;53:1560-9.
3. Breedveld FC, Weisman MIL Kavanaugh AF, et at. The PREMIER study: a multicenter, randomized, double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus methotrexate alone or adalimumab alone in patients with early, aggressive rheumatoid arthritis who had not had previous methotrexate treatment. Arthritis Rheum 2006;54:26-37.
4. Kievit W, Fransen J, Adang EMM, et at. Long-term effectiveness and safety of TNF-blocking agents in daily clinical practive: results from the Dutch rheumatoid arthritis monitoring register. Rheumatology (Oxford) 2011;50:196-203.
5. Bandres Ciga S, Salvatierra J, Lopez-Sidro M, et al. An examination of the mechanisms involved in secondary clinical failure to adalimumab or etanercept in
17 inflammatory arthropathies. J Clin Rheumatol 2015;21:115-9.
6. Radstake TR, Svenson M, Eijsbouts AM, et al. Formation of antibodies against infliximab and adalimumab strongly correlates with functional drug levels and clinical response in rheumatoid arthritis. Ann Rheum Dis 2009;68:1739-45.
7. van schouwenburg PA, van de Stadt LA, de Jong RN, et al. Adalimumab elicits a restricted anti-idiotypic antibody response in autoimmune patients resulting in functional neutralization. Ann Rheum Dis 2013;72:104-109.
8. Bartelds GM, Krieckaert CL, Nurmohamed MT, et al. Development of antidrug antibodies against adalimumab and association with disease activity and treatment failure during long-term follow-up. JAMA 2011;305:1460-68.
9. Balsa A, Sanmarti R, Rosas J, et al. Drug immunogenicity in patients with inflammatory arthritis and secondary failure to tumor necrosis factor inhibitor therapies: the REASON study. Rheurnatology (Oxford) 2018;57:688-93.
10. Chen DY, Chen YM, Tsai WC, et al. Significant associations of antidrug antibody levels with serum drug trough levels and therapeutic response of adalimumab and etanercept treatment in rheumatoid arthritis. Ann Rhettill Dis 2015;74:e16.
11. Smolen JS, Breedveld FC, Burmester GR, et al. Treating rheumatoid arthritis to target: 2014 update of the recommendations of an international task force. Ann Rheum Dis 2016;75:3-15.
12. Ortea I, Roschitzki B, Lopez-Rodriguez R, et al. Independent candidate serum protein biomarkers of response to adalimumab and to infliximab in rheumatoid arthritis: an exploratory study. PLoS One 2016;11:e0153140.
13. Cuppen B, Fritsch-Stork R, Eekhout I, et al. Proteomics to predict the response to tumor necrosis factor-alpha inhibitors in rheumatoid arthritis using a supervised
18 cluster-analysis based protein score. S'cand J Rheumatol 2018;47:12-21.
14. Tasaki S, Suzuki K, Kassai Y, et al. Multi-omics monitoring of drug response in rheumatoid arthritis in pursuit of molecular remission. Nat Commun 2018;9:2755.
15. Van Gestel AM, Prevoo ML, van't Hof MA, et al. Development and validation of the European League Against Rheumatism response criteria for rheumatoid arthritis.
Comparison with the preliminary American College of Rheumatology and the World Health Organization/International League Against Rheumatism Criteria.
Arthritis Rheum 1996;39:34-40.
16. Duarte J, Serufuri J-M, Mulder N, Blackburn J. Protein function microarrays: design, use and bioinformatic analysis in cancer biomarker discovery and quantitation.
In:
Wang X, editor. Bioinformatics of Human Proteomics. Dordrecht: Springer 2013;39-74.
17. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on bias and variance.
Bioinformatics 2003;19:185-193.
18. Johnson, WE, Rabinovic, A, and Li, C (2007). Adjusting batch effects in microarray expression data using Empirical Bayes methods. Biostatistics 8(1):118-127.
19. Bommert, A., Sun, X., Bischl, B., Rahnenfuhrer, J., Lang, M., 2020.
Benchmark for filter methods for feature selection in high-dimensional classification data.
Comput.
Stat. Data Anal. 143,106839.
20. Breiman, L., 2001. Random forests. Mach. Learn. 45 (1), 5-32.
21. Wright, M.N., Ziegler, A., 2017. Ranger: A fast implementation of random forests for high dimensional data in C++ and R. J. Stat. Softw. 77 (1), 1-17.
22. Kuhn, M.. Caret package. Journal of Statistical Software, 2008; 28(5) Strobl, C., Boulesteix, A.-L., Kneib, T., Augustin, T., Zeileis, A., 2008.
Conditional variable importance for random forests. BMC Bioinformatics 9 (307).

co Table 1 kµ.) No of test Biomarkers in panel ROC Sens Spec oc antigen t=4 SSB,TROVE2,ZHX2,PPARD,SPANXN2,HNRNPA231,TR1B2,CEP55,SH3GL1,FN3K,PANK3' 0.821 ranger permutation 20 0.806 0.774 HPCALLTHRA,A1FM1,0DC1,RPS6KA4,EEF1D,KLF10,EPHA2,PRKAR1A
cn SSB,TROVE2,ZHX2,PPARD,SPANXN2,HNRNPA231,TRIB2,CEP55,SH3GL1,FN3K,PANK3' 0.835 ranger_pernnutation 21 0.839 0.774 CO
HPCALLTHRA,A1FM1,0DC1,RPS6KA4,EEF1D,KLF10,EPHA2,PRKAR1A,EAPP
(r) SSB,TROVE2,ZHX2,PPARD,SPANXN2,HNRNPA2B1,TRIB2,CEP55,SH3G11,FN3K,PANK3, =I ranger_permutation 28 HPCALLTHRA,A1FM1,0DC1,RPS6KA4,EEF1D,KLF10,EPHA2,PRKAR1A,EAPP,ZNF331,G 0.824 0.839 0.710 MPS,POTEE,ASPSCR1,EC12,ETV7,BUD31 SSB,TROVE2,ZHX2,PPARD,SPANXN2,HNRNPA281,TRIB2,CEP55,SH3GL1,FN3K,PANK3, ranger_peramtation 29 HPCALLTHRA,A1FM1,0DC1,RPS6KA4,EEF1D,KLF10,EPHA2,PRKAR1A,EAPP,ZNF331,G
0.811 0.839 0.774 MPS,POTEE,ASPSCR1,EC12,ETV7,BUD31,ATF3 N-) (3) E.)) JI

Table 2 test Importance value Ranking SSB 0.003172 1 TROVE2 0.003142 2 ZHX2 0.002135 3 PPARD 0.001290 4 SPANXN2 0.000886 5 HNRNPA2B1 0.000870 6 TRIB2 0.000825 7 CEP55 0.000814 8 SH3GL1 0.000776 9 FN3K 0.000759 10 PANK3 0.000703 11 HPCAL1 0.000689 12 THRA 0.000657 13 AI FM1 0.000639 14 ODC1 0.000638 15 RPS6KA4 0.000613 16 EEF1D 0.000590 17 KLF10 0.000588 18 EPHA2 0.000587 19 PRKAR1A 0.000587 20 EAPP 0.000565 21 Table 3 Protein Name UniprotID Description SSB P05455 Lupus La protein Nucleotide Sequence (Seq ID No. 1):
>P003196_0311_0311Jube_SSB/La_6741_0_NM_003142.3_0_P05455_0 ATGGCTGAGAACGGCGACAACGAGAAGATGGCTGCTCTCGAGGCTAAGATCTGCCACCAGATCGAGTACTA
CTTGGGCGACTTGAACCTGCCGCGTGACAAGTTCGTGAAGGAACAGATGAAGGTGGAGGAGGGCTGGGTGC
CCCTCGAGATCATGATCAAGTTCAACCGTCTGAACCGCCTGACCACCGACTTCAACGTGATCGTCGAGGCTC
TGTCCAAGTCCAAGGCTGAGCTGATGGAAATCTCCGAGGACAAGACCAAGATCCGTCGTTCCCCATCCAAG
CCCCTGCCCGAAGTGACCGACGAGTACAAGAACGACGTGAAGAACCGTTCCGTGTACATCAAGGGTTTCCC
CACCGACGCTACCCTGGACGACATCAAGGAATGGCTCGAGGACAAGGGCCAGGTGCTGAACATCCAGATGC
GTCGTACCCTGCACAAGGCTTTCAAGGGTTCCATCTTCGTGGTGTTCGACTCCATCGAGTCCGCTAAGAAGT
TCGTCGAGACTCCCGGCCAGAAGTACAAGGAAACCGACCTGCTGATCCTGTTCAAGGACGACTACTTCGCC
AAGAAGAACGAGGAACGCAAGCAGAACAAGGTCGAGGCCAAGCTGCGTGCTAAGCAAGAGCAAGAGGCTA
AGCAGAAGCTGGAAGAGGACGCTGAGATGAAGTCCCTGGAAGAGAAGATCGGTTGCCTGCTGAAGTTCTCC
GGCGACCTGGACGACCAGACCTGCCGCGAGGACCTGCACATCCTGTTCTCCAACCACGGCGAGATCAAGT
GGATCGACTTCGTGCGTGGTGCTAAGGAAGGCATCATCCTCTTCAAGGAAAAGGCCAAGGAAGCTCTGGGC
AAGGCTAAGGACGCTAACAACGGCAACCTGCAGCTGCGTAACAAGGAAGTGACCTGGGAGGTCCTCGAGG
GCGAGGTCGAGAAGGAAGCCCTGAAGAAGATCATCGAGGACCAGCAAGAGTCCCTGAACAAGTGGAAGTCC
AAGGGTCGTCGTTTCAAGGGCAAGGGAAAGGGCAACAAGGCTGCTCAGCCCGGTTCCGGAAAGGGAAAGG
TGCAGTTCCAGGGCAAGAAGACCAAGTTCGCTTCCGACGACGAGCACGATGAGCACGACGAGAACGGTGCT
ACCGGTCCCGTGAAGCGTGCTCGTGAAGAGACTGACAAGGAAGAACCCGCTTCCAAGCAGCAAAAGACCGA
GAACGGCGCTGGCGACCAG
Protein Sequence (Seq ID No. 22):
>sp1P054551LA_HUMAN Lupus La protein OS=Homo sapiens OX=9606 GN=SSB PE=1 SV=2 MAENGDNEKMAALEAKICHQI EYYFGDFNLPRDKFLKEQI
KLDEGWVPLEIMIKFNRLNRLTTDFNVIVEALSKSKA
ELMEISEDKTKI RRSPSKPLPEVTDEYKN DVKN RSVYIKG FPTDATLD DI
KEWLEDKGQVLNIQMRRTLHKAFKGSI
FVVFDSIESAKKFVETPGQKYKETDLLI LFKDDYFAKKN EERKQNKVEAKLRAKQEQEAKQKLEEDAEMKSLEEKI

NLQLRNKEVTWEVL
EGEVEKEALKKII EDQQESLNKWKSKGRRFKGKGKGN KAAQPGSGKGKVQFQGKKTKFASDDEHDEHDENGAT
G PVKRAR EETDKEEPASKQQKTENGAG DO
TROVE2 P10155 HUMAN 60 kDa SS-A/Ro ribonucleoprotein Nucleotide Sequence (Seq ID No. 2):
>P001236_CAG_CAGp1_SSA2_6738_Homo sapiens Sjogren syndrome antigen A2 (60kDa ribonucleoprotein autoantigen SS-A/Ro)_BC036658.2_AAH36658.1_P10155_0_0_1617_0_1614 ATGGAGGAATCTGTAAACCAAATGCAGCCACTGAATGAGAAGCAGATAGCCAATTCTCAGGATGGATATGTA
TGGCAAGTCACTGACATGAATCGACTACACCGGTTCTTATGTTTCGGTTCTGAAGGTGGGACTTATTATATCA
AAGAACAGAAGTTGGGCCTTGAAAATGCTGAAGCTTTAATTAGATTGATTGAAGATGGCAGAGGATGTGAAG
TGATACAAGAAATAAAGTCATTTAGTCAAGAAGGCAGAACCACAAAGCAAGAGCCTATGCTCTTTGCACTTGC
CATTTGTTCCCAGTGCTCCGACATAAGCACAAAACAAGCAGCATTTAAAGCTGTTTCTGAAGTTTGTCGCATT
CCTACCCATCTCTTTACTTTTATCCAGTTTAAGAAAGATCTGAAGGAAAGCATGAAATGTGGCATGTGGGGTC
GTGCCCTCCGGAAGGCTATAGCGGACTGGTACAATGAGAAAGGTGGCATGGCCCTTGCTCTGGCAGTTACA
AAATATAAACAGAGAAATGGCTGGTCTCACAAAGATCTATTAAGATTGTCACATCTTAAACCTTCCAGTGAAG
GACTTGCAATTGTGACCAAATATATTACAAAGGGCTGGAAAGAAGTTCATGAATTGTATAAAGAAAAAGCACT
CTCTGTGGAGACTGAAAAATTATTAAAGTATCTGGAGGCTGTAGAGAAAGTGAAGCGCACAAGAGATGAGCT
AGAAGTCATTCATCTAATAGAAGAACATAGATTAGTTAGAGAACATCTTTTAACAAATCACTTAAAGTCTAAAG
AGGTATGGAAGGCTTTGTTACAAGAAATGCCGCTTACTGCATTACTAAGGAATCTAGGAAAGATGACTGCTAA
TTCAGTACTTGAACCAGGAAATTCAGAAGTATCTTTAGTATGTGAAAAACTGTGTAATGAAAAACTATTAAAAA
23 AGGCTCGTATACATCCATTTCATATTTTGATCGCATTAGAAACTTACAAGACAGGTCATGGTCTCAGAGGGAA
ACTGAAGTGGCGCCCTGATGAAGAAATTTTGAAAGCATTGGATGCTGCTTTTTATAAAACATTTAAGACAGTT
GAACCAACTGGAAAACGTTTCTTACTAGCTGTTGATGTCAGTGCTTCTATGAACCAAAGAGTTTTGGGTAGTA
TACTCAACGCTAGTACAGTTGCTGCAGCAATGTGCATGGTTGTCACACGAACAGAAAAAGATTCTTATGTAGT
TGCTTTTTCCGATGAAATGGTACCATGTCCAGTGACTACAGATATGACCTTACAACAGGTTTTAATGGCTATG
AGTCAGATCCCAGCAGGTGGAACTGATTGCTCTCTTCCAATGATCTGGGCTCAGAAGACAAACACACCTGCT
GATGTCTTCATTGTATTCACTGATAATGAGACCTTTGCTG GAG GTGTCCATCCTGCTATTGCTCTG AG G GAGT

ATCGAAAGAAAATGGATATTCCAGCTAAATTGATTGTTTGTGGAATGACATCAAATGGTTTCACCATTGCAGA
CCCAGATGATAGAGGCATGTTGGATATGTGCGGCTTTGATACTGGAGCTCTGGATGTAATTCGAAATTTCACA
TTAGATATGATT
Protein Sequence (Seq ID No. 23):
>spIP101551R060_HUMAN 60 kDa SS-A/Ro ribonucleoprotein OS=Homo sapiens OX=9606 GN=R060 PE=1 SV=2 MEESVNQMQPLNEKQIANSQDGYVWQVTDMNRLHRFLCFGSEGGTYYIKEQKLGLENAEALIRLIEDGRGCEVIQ
E I KSFSQEG RTTKQEPM LFALAICSQCS D ISTKQAAFKAVSEVC RI PTH LFTFIQ FKKDLKESM
KCGMWG RALR KAI
ADWYNEKGGMALALAVTKYKQRNGWSH KD LLR LS HLKPSSEG LAI VTKYITKGWKEVH
ELYKEKALSVETEKLLK
YLEAVEKVKRTRDELEVIHLIEEHRLVREHLLTNHLKSKEVWKALLQEMPLTALLRNLGKMTANSVLEPGNSEVSL
VC EKLC NEKLLK KAR I H PFH I LIALETYKTG H GLRGKLKWR P DE E I
LKALDAAFYKTFKTVEPTG KR FLLAVDVSASM

KTNTPADVFIVFTDN ETFAGGVH PAIALREYRKKM DI PAKLIVCGMTSNGFTIADPDDRGM LDMCG
FDTGALDVI R
NFTLDMI
ZHX2 09Y6X8 Zinc fingers and homeoboxes protein 2 Nucleotide Sequence (Seq ID No. 3):
> P002188 Q305 Q305p3 ZHX2 22882 Homo sapiens zinc fingers and homeoboxes 2 BC042145.1 Q9Y6X8 ATGGCTAGCAAAC GAAAATCTACAACTCCATG CAT GGTTCG G ACATCACAAGTAGTAG AACAAG
ATGTGCCC
GAGGAAGTAGACAGGGCCAAAGAGAAAGGAATCGGCACACCACAGCCTGACGTGGCCAAGGACAGTTGGG
CAGCAGAACTTGAAAACTCTTCCAAAGAAAACGAAGTGATAGAGGTGAAATCTATGGG GGAAAGCCAGTCCA
AAAAACTCCAAGGTGGTTATGAGTGCAAATACTGCCCCTACTCCACGCAAAACCTGAACGAGTTCACGGAGC
ATGTCGACATGCAGCATCCCAACGTGATTCTCAACCCCCTCTACGTGTGTGCAGAATGTAACTTCACAACCAA
AAAGTACGACTCCCTATCCGACCACAACTCCAAGTTCCATCCCGGGGAGGCCAACTTCAAGCTGAAGTTAAT
TAAACGCAATAATCAAACTGTCTTGGAACAGTCCATCGAAACCACCAACCATGTCGTGTCCATCACCACCAGT
GGCCCTGGAACTGGTGACAGTGATTCTGGGATCTCGGTGAGTAAAACCCCCATCATGAAGCCTGGAAAACC
AAAAGCGGATGCCAAGAAGGTGCCCAAGAAGCCCGAGGAGATCACCCCCGAGAACCACGTGGAAGGGACC
GCCCGCCTGGTGACAGACACAGCTGAGATCCTCTCGAGACTCGGCGGGGTGGAGCTCCTCCAAGACACATT
AG GACAC GTCATGCCTTCTGTACAGCTGCCACCAAATATCAACCTTGTGCCCAAG GTCCCTGTCCCACTAAA
TACTACCAAATACAACTCTGCCCTGGATACAAATGCCACGATGATCAACTCTTTCAACAAGTTTCCTTACCCG
ACCCAGGCTGAGTTGTCCTGGCTGACAGCTGCCTCCAAACACCCAGAGGAGCACATCAGAATCTGGTTTGC
CACCCAGCGCTTAAAGCATGGCATCAGCTGGTCCCCAGAAGAGGTGGAGGAGGCCCGGAAGAAGATGTTCA
ACGGCACCATCCAGTCAGTACCCCCGACCATCACTGTGCTGCCCGCCCAGTTGGCCCCCACAAAGGTGACG
CAGCCCATCCTCCAGACGGCTCTACCGTGCCAGATCCTCGGCCAGACTAGCCTG GTGCTGACTCAGGTGAC
CAGCGG GTCAACAACCGTCTCTTGCTCCCCCATCACACTTGCCGTG GCAG GAGTCACCAACCATG GCCAG A
AGAGACCCTTGGTGACTCCCCAAGCTGCCCCCGAACCCAAGCGTCCACACATCGCTCAGGTGCCAGAGCCC
CCACCCAAGGTGGCCAACCCCCCGCTCACACCAGCCAGTGACCGCAAGAAGACAAAGGAGCAGATAGCAC
ATCTCAAGGCCAGCTTTCTCCAGAGCCAGTTCCCTGACGATGCCGAGGTTTACCGGCTCATCGAGGTGACT
GGCCTTGCCAGGAGCGAGATCAAGAAGTGGTTCAGTGACCACCGATATCGGTGTCAAAGGGGCATCGTCCA
CATCACCAGCGAATCCCTTGCCAAAGACCAGTTGGCCATCGCGGCCTCCCGACACGGTCGCACGTATCATG
CGTACCCAGACTTTGCCCCCCAGAAGTTCAAAGAGAAAACACAGGGTCAGGTTAAAATCTTGGAAGACAGCT
TTTTGAAAAGTTCTTTTCCTACCCAAGCAGAACTGGATCGGCTAAGGGTGGAGACCAAGCTGAGCAGGAGAG
AGATCGACTCCTGGTTCTCGGAGAGGCGGAAGCTTCGAGACAGCATGGAACAAGCTGTCTTGGATTCCATG
GGGTCTGGCAAAAAAGGCCAAGATGTGGGAGCCCCCAATGGTGCTCTGTCTCGACTCGACCAGCTCTCCGG
TGCCCAGTTAACAAGTTCTCTGCCCAGCCCTTCGCCAGCAATTGCAAAAAGTCAAGAACAGGTTCATCTCCT
GAGGAGCACGTTTGCAAGAACCCAGTGGCCTACTCCCCAGGAGTACGACCAGTTAGCGGCCAAGACTGGCC
24 TGGTCCGAACTGAGATTGTGCGTTGGTTCAAGGAGAACAGATGCTTGCTGAAAACGGGAACCGTGAAGTGG
ATGGAGCAGTACCAGCACCAGCCCATGGCAGATGATCACGGCTACGATGCCGTAGCAAGGAAAGCAACAAA
ACCCATGGCCGAGAGCCCAAAGAACGGGGGTGATGTGGTTCCACAATATTACAAGGACCCCAAAAAGCTCT
GCGAAGAGGACTTGGAGAAGTTGGTGACCAGGGTAAAAGTAGGCAGCGAGCCAGCAAAAGACTGTTTGCCA
GCAAAGCCCTCAGAGGCCACCTCAGACCGGTCAGAGGGCAGCAGCCGGGACGGCCAGGGTAGCGACGAG
AACGAGGAGTCGAGCGTTGTGGATTACGTGGAGGTGACGGTCGGGGAGGAGGATGCGATCTCAGATAGAT
CAGATAGCTGGAGTCAGGCTGCGGCAGAAGGTGTGTCGGAACTGGCTGAATCAGACTCCGACTGCGTCCCT
GCAGAGGCTGGCCAGGCC
Protein Sequence (Seq ID No. 24):
>splQ9Y6X81ZHX2_HUMAN Zinc fingers and homeoboxes protein 2 OS=Homo sapiens OX=9606 GN=ZHX2 PE=1 SV=1 MASKRKSTTPCMVRTSQVVEQDVPEEVDRAKEKGIGTPQPDVAKDSWAAELENSSKENEVIEVKSMGESQSKKL
QGGYEC KYCPYSTQ N LNE FTEHVDMQHPNVILN PLYVCAECN FTTKKYDS LSDHN SKFH PG
EANFKLKLIKRNNO
TVLEQSIETTN HVVSITTSGPGTGDSDSGISVSKTPIMKPGKPKADAKKVPKKP
EEITPENHVEGTARLVTDTAEILS
RLGGVELLQDTLG HVM PSVQLPPN I NLVPKVPVPLNTTKYNSALDTNATMI
NSFNKFPYPTQAELSWLTAASKHPE
EH IRIWFATQRLKHGI SWSP EEVEEAR KKM EN GTIQSVP PTITVL FAQ LAPTKVTQPILQTALPCQILG
QTSLVLTQV
TSGSTTVSCSPITLAVAGVTNHGQKRPLVTPQAAPEPKRPHIAQVPEPPPKVANPPLTPASDRKKTKEQIAHLKAS
FLQSQFPDDAEVYRLIEVTGLARSEI KKWFSDH RYRCQRG IVH ITSESLAKDQLAIAASRHG
RTYHAYPDFAPQKF
KEKTQGQVKILEDSFLKSSFPTQAELDRLRVETKLSRREIDSWFSERRKLRDSMEQAVLDSMGSGKKGQDVGAP
NGALSRLDQLSGAQLTSSLPSPSPAIAKSQEQVHLLRSTFARTQWPTPQEYDOLAAKTGLVRTEIVRWFKENRCL
LKTGTVKWMEQYQHQPMADDHGYDAVARKATKPMAESPKNGGDVVPQYYKDPKKLCEEDLEKLVTRVKVGSE
PAKDCLPAKPSEATSDRSEGSSRDGQGSDENEESSVVDYVEVTVGEEDAISDRSDSWSQAAAEGVSELAESDS
DCVPAEAGQA
PPARD 003181 Peroxisome proliferator-activated receptor delta Nucleotide Sequence (Seq ID No. 4):
>P000475_SIG_SIG1-2_PPARD_5467_Homo sapiens peroxisome proliferative activated receptor delta transcript variant 2_BC002715.2_AAH02715.1_003181_51381.84_0_1086_0_1083 ATGGAGCAGCCACAGGAGGAAGCCCCTGAGGTCCGGGAAGAGGAGGAGAAAGAGGAAGTGGCAGAGGCA
GAAGGAGCCCCAGAGCTCAATGGGGGACCACAGCATGCACTTCCTTCCAGCAGCTACACAGACCTCTCCC
GGAGCTCCTCGCCACCCTCACTGCTGGACCAACTGCAGATGGGCTGTGACGGGGCCTCATGCGGCAGCCT
CAACATGGAGTGCCGGGTGTGCGGGGACAAGGCATCGGGCTTCCACTACGGTGTTCATGCATGTGAGGGG
TGCAAGGGCTTCTTCCGTCGTACGATCCGCATGAAGCTGGAGTACGAGAAGTGTGAGCGCAGCTGCAAGAT
TCAGAAGAAGAACCGCAACAAGTGCCAGTACTGCCGCTTCCAGAAGTGCCTGGCACTGGGCATGTCACACA
ACGCTATCCGTTTTGGTCGGATGCCGGAGGCTGAGAAGAGGAAGCTGGTGGCAGGGCTGACTGCAAATGA
GGGGAGCCAGTACAACCCACAGGTGGCCGACCTGAAGGCCTTCTCCAAGCACATCTACAATGCCTACCTGA
AAAACTTCAACATGACCAAAAAGAAGGCCCGCAGCATCCTCACCGGCAAAGCCAGCCACACGGCGCCCTTT
GTGATCCACGACATCGAGACATTGTGGCAGGCAGAGAAGGGGCTGGTGTGGAAGCAGTTGGTGAATGGCC
TGCCTCCCTACAAGGAGATCAGCGTGCACGTCTTCTACCGCTGCCAGTGCACCACAGTGGAGACCGTGCG
GGAGCTCACTGAGTTCGCCAAGAGCATCCCCAGCTTCAGCAGCCTCTTCCTCAACGACCAGGTTACCCTTC
TCAAGTATGGCGTGCACGAGGCCATCTTCGCCATGCTGGCCTCTATCGTCAACAAGGACGGGCTGCTGGTA
GCCAACGGCAGTGGCTTTGTCACCCGTGAGTTCCTGCGCAGCCTCCGCAAACCCTTCAGTGATATCATTGA
GCCTAAGTTTGAATTTGCTGTCAAGTTCAACGCCCTGGAACTTGATGACAGTGACCTGGCCCTATTCATTGC
GGCCATCATTCTGTGTGGAGGTGAG
Protein Sequence (Seq ID No. 25):
>sp10031811PPARD_HUMAN Peroxisome proliferator-activated receptor delta OS=Homo sapiens OX=9606 GN=PPARD PE=1 SV=1 MEQPQEEAPEVREEEEKEEVAEAEGAPELNGGPQHALPSSSYTDLSRSSSPPSLLDQLQMGCDGASCGSLNM
ECRVCGDKASGFHYGVHACEGCKGFFRRTIRMKLEYEKCERSCKIQKKNRNKCQYCRFQKCLALGMSHNAI RF
G RM PEAEKRKLVAGLTAN EGSQYN PQVADLKAFSKH IYNAYLKN FNMTKKKARSI LTGKASHTAPFVIH
DI ETLW
QAEKGLVWKOLVNGLPPYKEISVHVFYRCQCTTVETVRELTEFAKSIPSFSSLFLNDQVTLLKYGVHEAIFAMLASI

VN KDG LLVANGSG FVTREFLRSLRKP FSDI I EPKFEFAVKFNALELDDSDLALFIAAI I LCGDRPG LM
NVP RVEAIQ D
TI LRALEFH LOAN H P DAQYLFPKLLQKMADLROLVTEHAQMMQR1 KKTETETSLHP LLQ ElYKDMY
SPANXN2 Q5MJ10 Sperm protein associated with the nucleus on the X chromosome N2 Nucleotide Sequence (Seq ID No. 5):
>P003098_0211_0211 Jube_SPANXN2_494119_0_NM_001009615.1_0_05MJ10_0_Insert sequence is gene optimized by GeneArt_0_0_0 ATGGAACAGCCCACCTCTTCCACCAACGGCGAGAAGCGCAAGTCCCCCTGCGAGTCCAACAACAAGAAAAA
CGACGAGATGCAAGAGGCTCCCAACCGTGTGCTGGCTCCCAAGCAGTCCCTGCAAAAGACCAAGACCATC
GAGTACCTGACCATCATCGTGTACTACTACCGCAAGCACACCAAGATCAACTCCAACCAGCTCGAGAAGGA
CCAGTCCCGCGAGAACTCCATCAACCCCGTGCAAGAGGAAGAGGACGAGGGCCTGGACTCCGCTGAGGG
ATCCTCCCAAGAAGATGAGGACCTGGACAGCTCCGAGGGTTCCAGCCAAGAGGATGAAGATCTCGACTCCT
CCGAGGGCAGCTCCCAAGAGGACGAGGACTTG GATTCCTCCGAGGGATCTAGTCAAGAGGACGAGGATCT
GGACTCTTCCGAAGGCTCATCTCAAGAAGATGAAGATTTGGACCCCCCTGAGGGTAGCAGTCAAGAGGATG
AGGACCTCGATTCCAGCGAGGGCTCCTCACAAGAGGGTGGCGAGGAT
Protein Sequence (Seq ID No. 26):
>splQ5MJ101SPXN2 HUMAN Sperm protein associated with the nucleus on the X
chromosome N2 OS=Homo sapiens OX=9606 GN=SPANXN2 PE=1 SV=1 M EQPTSSTNGEKRKSPCESN NKKN DEMQEAP N RVLAPKQSLQKTKTI EYLTI I VYYYRKHTKINSNQ
LEKDQSRE
NSINPVQEEEDEGLDSAEGSSQEDEDLDSSEGSSOEDEDLDSSEGSSQEDEDLDSSEGSSQEDEDLDSSEGSS
Q EDED LDP P EG SSQ ED ED LDSS EG SSQ EGG ED
HN RN PA2 B1 P22626 Heterogeneous nuclear ribonucleoproteins Nucleotide Sequence (Seq ID No. 6):
>P003186 0311 0311 tube HNRNPA2B1 3181 0 NM 002137.3 0 P22626 0 ATGGAACGCGAGAAAGAGCAGTTCCGCAAGCTGTTCATCGGTGGCCTGTCCTTCGAGACTACCGAGGAATC
CCTGCGCAACTACTACGAGCAGTGGGGCAAGCTGACCGACTGCGTGGTCATGCGTGACCCCGCTTCCAAG
CGTTCCCGTGGTTTCGGTTTCGTGACCTTCTCCAGCATGGCTGAGGTGGACGCTGCTATGGCTGCTCGTCC
CCACTCCATCGACGGTCGTGTGGTCGAGCCTAAG CGTGCTGTGGCTCGTGAAGAGTCCGGCAAGCCTG GT
GCTCACGTGACCGTGAAGAAGCTGTTCGTTGGCG GTATCAAAGAGGACACCGAG GAACACCACCTGAGGG
ACTACTTCGAGGAATACGGCAAGATCGACACCATCGAGATCATCACCGACCGTCAGTCCGGAAAGAAGCGC
GGCTTCGGCTTCGTCACTTTCGACGACCACGACCCCGTGGACAAGATCGTGCTGCAGAAGTACCACACCAT
CAACGGTCACAACGCTGAAGTGCGCAAGGCTCTGTCCCGTCAAGAGATGCAAGAGGTGCAGTCCTCCCGTT
CCGGTCGTGGTGGCAACTTCGGATTCGGCGACTCTCGCGGTGGTG GCGG AAACTTCGGTCCTGGTCCCGG
TTCCAACTTCCGTGGTGGTTCCGACGGTTACGGCTCCGGAAGAGGTTTCGGCGACGGCTACAACGGCTAC
GGTGGTGGTCCTGGCGGTGGAAATTTCGGTGGTTCCCCTGGTTACGGTGGCGGTCGCGGTGGATACGGCG
GAGGTGGTCCAGGATACGGCAACCAGGGTGGCGGTTACGGCGGTGGTTACGACAACTACGGTGGCGGCA
ACTACGGTTCCGGAAACTACAACGACTTCGGCAATTACAACCAGCAGCCCTCCAACTACGGCCCCATGAAG
TCTGGCAATTTCGGCGGCTCCCGTAACATGGGTGGTCCTTACGGTGGTGGAAATTACGGTCCCGGTGGTTC
CGGTGGCTCTGGTGGCTACGGCGGTCGTTCCCGTTAC
Protein Sequence (Seq ID No. 27):
>spIP226261ROA2_HUMAN Heterogeneous nuclear ribonucleoproteins A2/B1 OS=Homo sapiens OX=9606 GN=HNRNPA2B1 PE=1 SV=2 MEKTLETVPLERKKREKEQFRKLFIGGLSFETTEESLRNYYEQWGKLTDCVVMRDPASKRSRGFGFVTFSSMAE
VDAAMAARP HSI DG RVVEPKRAVAREESG KPGAHVTVKKLFVGGI KEDTEEHHLRDYFEEYGKI DTI El ITDRQSG
KKRGFGFVTFDDHDPVDKIVLQKYHTI NG H NAEVRKALSRQ EMQEVQSSRSG RGG N FG FG DSRGGGG
NFG PG
PGSNFRGGSDGYGSGRGFGDGYNGYGGGPGGGNFGGSPGYGGGRGGYGGGGPGYGNQGGGYGGGYDNY
GGGNYGSGNYNDFGNYNQQPSNYGPMKSG NFGGSRNMGGPYGGGNYGPGGSGGSGGYGGRSRY

TRIB2 092519 HUMAN Tribbles homolog 2 Nucleotide Sequence (Seq ID No. 7):
>R001066_K I N2_KI N2p1_TRB2_28951_Homo sapiens tribbles homolog 2_RC002637.2_AAH02637.1_092519_0_0_1032_0_1029 ATGAACATACACAGGTCTACCCCCATCACAATAGCGAGATATGGGAGATCGCGGAACAAAACCCAGGATTT
CGAAGAGTTGTCGTCTATAAGGTCCGCGGAGCCCAGCCAGAGTTTCAGCCCGAACCTCGGCTCCCCGAGC
CCGCCCGAGACTCCGAACTTGTCGCATTGCGTTTCTTGTATCGGGAAATACTTATTGTTGGAACCTCTGGAG
GGAGACCACGTTTTTCGTGCCGTGCATCTGCACAGCGGAGAGGAGCTGGTGTGCAAGGTGTTTGATATCAG
CTGCTACCAGGAATCCCTGGCACCGTGCTTTTGCCTGTCTGCTCATAGTAACATCAACCAAATCACTGAAAT
TATCCTGG GT G AGACCAAAGCCTATGTGTTCTTTG AGC GAAGCTATGG G
GACATGCATTCCTTCGTCCGCAC
CTGCAAGAAGCTGAGAGAGGAGGAGGCAGCCAGACTGTTCTACCAGATTGCCTCGGCAGTGGCCCACTGC
CATGACGGGGGGCTGGTGCTGCGGGACCTCAAGCTGCGGAAATTCATCTTTAAGGACGAAGAGAGGACTC
GGGTCAAGCTGGAAAGCCTGGAAGACGCCTACATTCTGCGG GGAGATGATGATTCCCTCTCCGACAAGCAT
GGCTGCCCGGCTTACGTAAGCCCAGAGATCTTGAACACCAGTGGCAGCTACTCGGGCAAAGCAGCCGACG
TGTGGAGCCTGGGGGTGATGCTGTACACCATGTTGGTGGGGCGGTACCCTTTCCATGACATTGAACCCAGC
TCCCTCTTCAGCAAGATCCGGCGTGGCCAGTTCAACATTCCAGAGACTCTGTCGCCCAAGGCCAAGTGCCT
CATCCGAAGCATTCTGCGTCGGGAGCCCTCAGAGCGGCTGACCTCGCAGGAAATTCTGGACCATCCTTGGT
TTTCTACAGATTTTAGCGTCTCGAATTCAGCATATGGTGCTAAGGAAGTGTCTGACCAGCTGGTGCCGGACG
TCAACATGGAAGAGAACTTGGACCCTTTCTTTAAC
Protein Sequence (Seq ID No. 28):
>splQ925191TRIB2 HUMAN Tribbles homolog 2 OS=Homo sapiens OX=9606 GN=TRIB2 PE=1 SV=1 M NI HRSTPITIARYGRSRNKTODFEELSSI RSAEPSOSFSPN LGSPSP P ET P N LSHCVSCI GKYL
LLEP LEGDHVFR
AVHLHSGEELVCKVFDISCYQESLAPCFCLSAHSNI NQITEI I LG ETKAYVFFERSYG DM
HSFVRTCKKLREEEAAR
LFYQIASAVAHCH DG GLVLR DLKLR KFI FKDEERTRVKLESLEDAYI LRG DDDSLSDKHGCPAYVSPEI
LNTSGSY
SGKAADVWSLGVMLYTM LVG RYPFHDI EPSSLFSKIRRGQFNI P ETLSP KAKC LI RSI LR RE PSE
RLTSQEI LDH PW
FSTDFSVSNSAYGAKEVSDQLVPDVNMEENLDPFFN
CEP55 053EZ4 Centrosomal protein of 55 kDa Nucleotide Sequence (Seq ID No. 8):
>P003121 0211 0211 tube CEP55 55165 0 NM 001127182.1 0 053EZ4 0 ATGTCCTCCCGTTCCACCAAGGACCTGATCAAGTCTAAGTGGGGTTCCAAGCCCTCCAACTCCAAGTCCGA
GACTACCCTCGAGAAGCTGAAGGGCGAGATCGCTCACCTCAAGACCTCCGTGGACGAGATCACCTCCGGC
AAGGGCAAGCTGACCGACAAGGAACGTCACCGTCTGCTCGAGAAGATCCGTGTGCTCGAGGCTGAGAAGG
AAAAGAACGCTTACCAGCTGACTGAGAAGGACAAGGAAATCCAGCGTCTGCGCGACCAGCTGAAGGCTCGT
TACTCCACCACCGCTCTGCTGGAACAGCTGGAAGAAACCACCCGCGAGGGCGAGCGTCGCGAGCAGGTCC
TGAAGGCTCTGTCCGAAGAGAAGGACGTGCTGAAGCAGCAGCTGTCCGCTGCTACCTCCCGTATCGCTGA
GCTGGAATCCAAGACCAACACCCTGCGTCTGTCCCAGACCGTGGCTCCCAACTGCTTCAACTCCTCCATCA
ACAACATCCACGAGATGGAAATCCAACTGAAGGACGCTCTCGAGAAGAACCAGCAGTGGCTGGTGTACGAC
CAGCAGCGCGAGGTGTACGTGAAGGGCCTGCTGGCTAAGATCTTCGAGCTGGAAAAGAAGACCGAGACTG
CTGCTCACTCCCTGCCCCAGCAGACCAAGAAGCCCGAGTCCGAGGGTTACCTGCAAGAGGAAAAGCAGAA
GTGCTACAACGACCTGCTGGCTTCCGCTAAGAAGGACCTGGAAGTCGAGCGTCAGACCATCACCCAGCTGT
CCTTCGAGCTGTCCGAGTTCCGTAGGAAGTACGAAGAGACTCAGAAGGAAGTCCACAACCTGAACCAGCTG
CTGTACTCCCAGCGTCGTGCTGACGTGCAGCACCTCGAGGACGACCGTCACAAGACTGAGAAGATCCAGA
AGCTGCGCGAAGAGAACGATATCGCTCGTGGCAAGCTCGAGGAAGAGAAGAAGCGTTCCGAGGAACTGCT
GTCCCAGGTGCAGTTCCTGTACACCTCCCTGCTCAAGCAGCAAGAGGAACAGACCCGTGTGGCTCTGTTGG
AGCAGCAGATGCAGGCTTGCACCCTGGACTTCGAGAACGAGAAGCTGGACCGTCAGCACGTCCAGCACCA
GCTGCACGTGATCCTGAAGGAACTGCGCAAGGCTCGTAACCAGATCACCCAGTTGGAGTCCCTGAAGCAG
CTGCACGAGTTCGCTATCACCGAGCCCCTGGTCACCTTCCAAGGCGAGACTGAGAACCGCGAGAAGGTGG
CCGCTTCCCCCAAGTCCCCCACCGCTGCTCTGAACGAGTCCCTGGTCGAGTGCCCCAAGTGCAACATCCA
GTACCCCGCTACCGAGCACCGTGACCTGCTGGTGCACGTCGAGTACTGCTCCAAG

Protein Sequence (Seq ID No. 29):
>splQ53EZ4ICEP55_HUMAN Centrosomal protein of 55 kDa OS=Homo sapiens OX=9606 GN=CEP55 PE=1 SV=3 MSSRSTKDLIKSKWGSKPSNSKSETTLEKLKGEIAHLKTSVDEITSGKGKLTDKERHRLLEKIRVLEAEKEKNAYQ
LTEKDKEIQRLRDQLKARYSTTTLLEQLEETTREGERREQVLKALSEEKDVLKQQLSAATSRIAELESKTNTLRLS
QTVAPNCFNSSI N NI HEM EIQ LKDALEKNQQWLVYDQQ REVYVKG LLAKI FELEK
KTETAAHSLPQQTKKP ESEG
YLQEEKQKCYNDLLASAKKDLEVEROTITQLSFELSEFRRKYEETQKEVHNLNQLLYSORRADVQHLEDDRHKT
EKIQKLREENDIARGKLEEEKKRSEELLSQVQFLYTSLLKQQEEQTRVALLEQQMQACTLDFENEKLDRQHVQH
QLHVILKELRKARNQITQLESLKQLHEFAITEPLVTFQGETENREKVAASPKSPTAALNESLVECPKCNIQYPATEH
RDLLVHVEYCSK
SH3GL1 099961 Endophilin-A2 Nucleotide Sequence (Seq ID No. 9):
>P000121_CAN_CAN1-1_SH3GL1_6455_Homo sapiens SH3-domain GRB2-like 1_BC001270.1_AAH01270.1_099961_0_0_1107_0_1104 ATGTCGGTGGCGGGGCTGAAGAAGCAGTTCTACAAGGCGAGCCAGCTGGTCAGTGAGAAGGTCGGAGGG
GCCGAGGGGACCAAGCTGGATGATGACTTCAAAGAGATGGAGAAGAAGGTGGATGTCACCAGCAAGGCGG
TGACAGAAGTGCTGGCCAGGACCATCGAGTACCTGCAGCCCAACCCAGCCTCGCGGGCTAAGCTGACCAT
GCTCAACACGGTGTCCAAGATCCGGGGCCAGGTGAAGAACCCCGGCTACCCGCAGTCGGAGGGGCTTCTG
GGCGAGTGCATGATCCGCCACGGGAAGGAGCTGGGCGGCGAGTCCAACTTTGGTGACGCATTGCTGGATG
CCGGCGAGTCCATGAAGCGCCTGGCAGAGGTGAAGGACTCCCTGGACATCGAGGTCAAGCAGAACTTCAT
TGACCCCCTCCAGAACCTGTGCGAGAAAGACCTGAAGGAGATCCAGCACCACCTGAAGAAACTGGAGGGC
CGCCGCCTGGACTTTGACTACAAGAAGAAGCGGCAGGGCAAGATCCCCGATGAGGAGCTACGCCAGGCGC
TGGAGAAGTTCGAGGAGTCCAAGGAGGTGGCAGAAACCAGCATGCACAACCTCCTGGAGACTGACATCGA
GCAGGTGAGTCAGCTCTCGGCCCTGGTGGATGCACAGCTGGACTACCACCGGCAGGCCGTGCAGATCCTG
GACGAGCTGGCGGAGAAGCTCAAGCGCAGGATGCGGGAAGCTTCCTCACGCCCTAAGCGGGAGTATAAGC
CGAAGCCCCGGGAGCCCTTTGACCTTGGAGAGCCTGAGCAGTCCAACGGGGGCTTCCCCTGCACCACAGC
CCCCAAGATCGCAGCTTCATCGTCTTTCCGATCTTCCGACAAGCCCATCCGGACCCCTAGCCGGAGCATGC
CGCCCCTGGACCAGCCGAGCTGCAAGGCGCTGTACGACTTCGAGCCCGAGAACGACGGGGAGCTGGGCT
TCCATGAGGGCGACGTCATCACGCTGACCAACCAGATCGATGAGAACTGGTACGAGGGCATGCTGGACGG
CCAGTCGGGCTTCTTCCCGCTCAGCTACGTGGAGGTGCTTGTGCCCCTGCCGCAG
Protein Sequence (Seq ID No. 30):
>splQ999611SH3G1_HUMAN Endophilin-A2 OS=Homo sapiens OX=9606 GN=SH3GL1 PE=1 SV=1 MSVAGLKKQFYKASQLVSEKVGGAEGTKLDDDFKEMEKKVDVTSKAVTEVLARTIEYLQPNPASRAKLTMLNTV
SKI RGQVKNPGYPQSEG LLGECMI RHG KELGG ESN FGDALLDAG ESM KRLAEVKDSLDI EVKQNFI
DPLQNLCEK
DLKEIQHHLKKLEGRRLDFDYKKKRQGKI PDEELRQALEKFEESKEVAETSMH NLLETDIEQVSQLSALVDAQLDY

H ROAM LDELAEKLKRRM REASSRP KREYKP KPREP FDLG EP EQSNGGFPCTTAP KIAASSSFRSSDKP
I RTPS
RSMP P LDQ PSCKALYDFEP EN DGELGFHEGDVITLTNQ I DENWYEGMLDGQSG FFPLSYVEVLVPLPQ
FN3K 09H479 Fructosamine-3-kinase Nucleotide Sequence (Seq ID No. 10):
>P002359 0106 Q106p1 FN3K 64122 Homo sapiens fructosamine 3 ki nase_BC042680.1_AAH 42680.1_09 H 479_0_0_930_0_927 ATGGAGCAGCTGCTGCGCGCCGAGCTGCGCACCGCGACCCTGCGGGCCTTCGGCGGCCCCGGCGCCGG
CTGCATCAGCGAGGGCCGAGCCTACGACACGGACGCAGGCCCAGTGTTCGTCAAAGTCAACCGCAGGACG
CAGGCCCGGCAGATGTTTGAGGGGGAGGTGGCCAGCCTGGAGGCCCTCCGGAGCACGGGCCTGGTGCG
GGTGCCGAGGCCCATGAAGGTCATCGACCTGCCGGGAGGTGGGGCCGCCTTTGTGATGGAGCATTTGAAG
ATGAAGAGCTTGAGCAGTCAAGCATCAAAACTTGGAGAGCAGATGGCAGATTTGCATCTTTACAACCAGAAG
CTCAGGGAGAAGTTGAAGGAGGAGGAGAACACAGTGGGCCGAAGAGGTGAGGGTGCTGAGCCTCAGTATG
TGGACAAGTTCGGCTTCCACACGGTGACGTGCTGCGGCTTCATCCCGCAGGTGAATGAGTGGCAGGATGA

CTGGCCGACCTTTTTCGCCCGGCACCGGCTCCAGGCGCAGCTGGACCTCATTGAGAAGGACTATGCTGAC
CGAGAGGCACGAGAACTCTGGTCCCGGCTACAGGTGAAGATCCCGGATCTGTTTTGTGGCCTAGAGATTGT
CCCCGCGTTGCTCCACGGGGATCTCTGGTCGGGAAACGTGGCTGAGGACGACGTGGGGCCCATTATTTAC
GACCCGGCTTCCTTCTATGGCCATTCCGAGTTTGAACTG GCAATCGCCTTGATGTTTGGGGGGTTCCCCAG
ATCCTTCTTCACCGCCTACCACCGGAAGATCCCCAAGGCTCCGGGCTTCGACCAGCGGCTGCTGCTCTACC
AGCTGTTTAACTACCTGAACCACTGGAACCACTTCGGGCGGGAGTACAGGAGCCCTTCGTTGGGCACCATG
CGAAGGCTGCTCAAG
Protein Sequence (Seq ID No. 31):
>splQ9H4791FN3K HUMAN Fructosamine-3-kinase OS=Homo sapiens OX=9606 GN=FN3K
PE=1 SV=1 MEOLLRAELRTATLRAFGGPGAGCISEGRAYDTDAGPVFVKVNRRTQARQMFEGEVASLEALRSTGLVRVPRP
MKVI DLPGGGAAFVMEHLKMKSLSSQASKLG EQMADLHLYNQKLREKLKEEENTVGRRG EGAEPQYVDKFGFH
TVTCCGFI PQVNEWQDDWPTFFARHRLQAQLDLIEKDYADREARELWSRLQVKI PDLFCGLEIVPALLHG DLWS
G NVAEDDVGPI IYDPASFYGHSEFELAIALM FGGFPRSFFTAYHRKI PKAPG FDQRLLLYQLFNYLN HWN
HFG RE
YRSPSLGTMRRLLK
PANK3 09H999 Pantothenate kinase 3 Nucleotide Sequence (Seq ID No. 11):
>P002239 0106 0106p2 PANK3 79646 Homo sapiens pantothenate kinase 3_BC013705.1_AAH13705.1_09H999_0_0_1113_0_1110 ATGAAGATCAAAGATGCCAAGAAACCCTCTTTCCCATGGTTTGGCATGGACATTGGGGGAACTCTAGTAAAG
CTCTCGTACTTTGAACCTATTGATATCACAGCAGAGGAAGAGCAAGAAGAAGTTGAGAGTTTAAAAAGTATTC
GGAAATATTTGACTTCTAACGTGGCATATGGATCCACCGGCATTCGGGATGTACACCTTGAACTGAAAGATT
TAACACTTTTTGGCCGAAGAGGGAACTTGCACTTTATCAGGTTTCCAACCCAGGACCTGCCTACTTTTATCCA
AATGGGAAGAGATAAAAACTTCTCAACATTGCAGACGGTGCTATGTGCTACAGGAGGTGGTGCTTACAAGTT
TGAAAAAGATTTTCGCACAATTGGAAACCTCCACCTGCACAAACTGGATGAACTTGACTGCCTTGTAAAGGG
CTTGCTGTATATAGACTCTGTCAGTTTCAATGGACAAGCCGAGTGCTATTATTTTGCTAATGCCTCAGAACCT
GAGCGATGCCAAAAGATGCCTTTTAACCTGGATGATCCCTATCCACTGCTTGTAGTGAACATTGGCTCAGGA
GTCAGTATTTTAGCAGTCCATTCCAAAGACAACTATAAACGAGTGACTGGGACAAGCCTTGGAGGGGGTAC
CTTTCTGGGTTTATGCAGTTTATTGACTGGCTGTGAAAGTTTTGAAGAGGCTCTTGAAATGGCATCCAAAGGT
GATAGCACACAAGCTGACAAGCTGGTCCGTGATATTTATGGAGGAGATTATGAAAGATTTGGTTTGCCAGGT
TGGGCTGTAGCATCTAGTTTTGGGAATATGATTTATAAGGAGAAGCGAGAATCTGTTAGTAAAGAAGATCTG
GCAAGAGCTACTTTAGTTACTATCACCAATAACATTGGTTCTGTGGCACGAATGTGTGCTGTTAATGAGAAAA
TAAACAGAGTTGTCTTTGTTGGAAACTTTTTACGTGTCAATACCCTCTCAATGAAACTTTTGGCATATGCACT
GGATTACTGGTCAAAAGGTCAACTAAAAGCATTGTTTCTAGAACATGAGGGTTACTTTGGAGCAGTTGGTGC
ACTTCTTGGGCTGCCAAATTTCAGC
Protein Sequence (Seq ID No. 32):
>splQ9H9991PANK3_HUMAN Pantothenate kinase 3 OS=Homo sapiens OX=9606 GN=PANK3 PE=1 SV=1 MKI KDAKKPSFPWFGMDIGGTLVKLSYFEPIDITAEEEQEEVESLKSI RKYLTSNVAYGSTG IR
DVHLELKDLTLFG
RRGNLHFI RFPTQDLPTFIQMGRDKNFSTLQTVLCATGGGAYKFEKDFRTIGNLHLHKLDELDCLVKGLLYI DSVS

FNGQAECYYFANASEPERCQKMPFNLDDPYPLLVVNIGSGVSILAVHSKDNYKRVTGTSLGGGTFLGLCSLLTGC
ESFEEALEMASKGDSTQADKLVRDIYGGDYERFGLPGWAVASSFGNMIYKEKRESVSKEDLARATLVTITNNIGS
VARMCAVNEKI NRVVFVGNFLRVNTLSMKLLAYALDYWSKGQLKALFLEHEGYFGAVGALLGLPNFS
H PCAL1 P37235 Hippocalcin-like protein 1 Nucleotide Sequence (Seq ID No. 12):
>P003172_0311_0311Jube_HPCAL1_3241_0_NM_002149.2_0_P37235_0 ATGGGCAAGCAGAACTCCAAGCTGCGTCCCGAGGTGCTGCAGGACCTGCGCGAGAACACCGAGTTCACCG
ACCACGAGCTGCAAGAGTGGTACAAGGGTTTCCTGAAGGACTGCCCCACCGGTCACCTGACCGTGGACGA
GTTCAAGAAGATCTACGCTAACTTCTTCCCCTACGGCGACGCTTCCAAGTTCGCTGAGCACGTGTTCCGTAC

CTTCGACACCAACGGCGACGGCACCATCGACTTCCGCGAGTTCATCATCGCTCTGTCCGTGACCTCCCGTG
GCAAGCTCGAGCAAAAGCTGAAGTGGGCTTTCTCGATGTACGACCTGGACGGCAACGGTTACATCTCCCGT
TCCGAGATGCTCGAGATCGTGCAG GCTATCTACAAGATGGTGTCCTCCGTGATGAAGATGCCCGAGGACGA
GTCCACCCCCGAGAAGCGTACCGACAAGATCTTCCGTCAGATGGACACCAACAACGACGGAAAGCTGTCCC
TGGAAGAGTTCATCCGTGGTGCTAAGTCCGACCCCTCCATCGTGCGTCTGCTGCAGTGCGACCCATCCTCC
GCTTCCCAGTTC
Protein Sequence (Seq ID No. 33):
>spIP372351HPCD_HUMAN Hippocalcin-like protein 1 OS=Homo sapiens OX=9606 GN=HPCAL1 PE=1 SV=3 MGKQNSKLRPEVLQDLRENTEFTDHELQEWYKGFLKDCPTGHLTVDEFKKIYANFFPYGDASKFAEHVFRTFDT
NGDGTI DFREFIIALSVTSRGKLEQKLKWAFSMYDLDGNGYISRSEMLEIVQAIYKMVSSVMKMPEDESTPEKRTD
KIFRQMDTNNDGKLSLEEFIRGAKSDPSIVRLLQCDPSSASQF
TH RA P10827 Thyroid hormone receptor alpha Nucleotide Sequence (Seq ID No. 13):
>P000757_TRN_TRNp2_THRA_7067_Homo sapiens Homo sapiens thyroid hormone receptor alpha (erythroblastic leukemia viral (v-erb-a) onc_BC000261.1_AAH00261.1_P10827_0_0_1473_0_1470 ATGGAACAGAAGCCAAGCAAGGTGGAGTGTGGGTCAGACCCAGAGGAGAACAGTGCCAGGTCACCAGATG
GAAAGCGAAAAAGAAAGAACGGCCAATGTTCCCTGAAAACCAGCATGTCAGGGTATATCCCTAGTTACCTG
GACAAAGACGAGCAGTGTGTCGTGTGTGGGGACAAGGCAACTGGTTATCACTACCGCTGTATCACTTGTGA
GGGCTGCAAGGGCTTCTTTCGCCGCACAATCCAGAAGAACCTCCATCCCACCTATTCCTGCAAATATGACA
GCTGCTGTGTCATTGACAAGATCACCCGCAATCAGTGCCAGCTGTGCCGCTTCAAGAAGTGCATCGCCGTG
GGCATGGCCATGGACTTGGTTCTAGATGACTCGAAGCGGGTGGCCAAGCGTAAGCTGATTGAGCAGAACC
GGGAGCGGCGGCGGAAGGAGGAGATGATCCGATCACTGCAGCAGCGACCAGAGCCCACTCCTGAAGAGT
GGGATCTGATCCACATTGCCACAGAGGCCCATCGCAGCACCAATGCCCAGGGCAGCCATTGGAAACAGAG
GCGGAAATTCCTGCCCGATGACATTGGCCAGTCACCCATTGTCTCCATGCCGGACGGAGACAAGGTGGAC
CTGGAAGCCTTCAGCGAGTTTACCAAGATCATCACCCCGGCCATCACCCGTGTGGTGGACTTTGCCAAAAA
ACTGCCCATGTTCTCCGAGCTGCCTTGCGAAGACCAGATCATCCTCCTGAAGGGGTGCTGCATGGAGATCA
TGTCCCTGCGGGCGGCTGTCCGCTACGACCCTGAGAGCGACACCCTGACGCTGAGTGGGGAGATGGCTGT
CAAGCGGGAGCAGCTCAAGAATGGCGGCCTGGGCGTAGTCTCCGACGCCATCTTTGAACTGGGCAAGTCA
CTCTCTGCCTTTAACCTGGATGACACGGAAGTGGCTCTGCTGCAGGCTGTGCTGCTAATGTCAACAGACCG
CTCGGGCCTGCTGTGTGTGGACAAGATCGAGAAGAGTCAGGAGGCGTACCTGCTGGCGTTCGAGCACTAC
GTCAACCACCGCAAACACAACATTCCGCACTTCTGGCCCAAGCTGCTGATGAAGGAGAGAGAAGTGCAGAG
TTCGATTCTGTACAAGGGGGCAGCGGCAGAAGGCCGGCCGGGCGGGTCACTGGGCGTCCACCCGGAAGG
ACAGCAGCTTCTCGGAATGCATGTTGTTCAGGGTCCGCAGGTCCGGCAGCTTGAGCAGCAGCTTGGTGAA
GCGGGAAGTCTCCAAGGGCCGGTTCTTCAGCACCAGAGCCCGAAGAGCCCGCAGCAGCGTCTCCTGGAG
CTGCTCCACCGAAGCGGAATTCTCCATGCCCGAGCGGTCTGTGGGGAAGACGACAGCAGTGAGGCGGACT
CCCCGAGCTCCTCTGAGGAGGAACCGGAGGTCTGCGAGGACCTGGCAGGCAATGCAGCCTCTCCC
Protein Sequence (Seq ID No. 34):
>spIP108271THA HUMAN Thyroid hormone receptor alpha OS=Homo sapiens OX=9606 GN=THRA PE=1 SV=1 MEQKPSKVECGSDPEENSARSPDGKRKRKNGQCSLKTSMSGYI PSYLDKDEQCVVCGDKATGYHYRCITCEGC
KGFFRRTIQKNLHPTYSCKYDSCCVI DKITRNQCQLCRFKKCIAVGMAMDLVLDDSKRVAKRKLI EQNRERRRKE
EMI RSLQQRPEPTPEEWDLI H IATEAH RSTNAQGSHWKQRRKFLP DDI GQS PI VSM P DG DKVD
LEAFSEFTKI ITP
AITRVVDFAKKLPMFSELPCEDQI I LLKGCCMEI MSLRAAVRYDP ESDTLTLSG EMAVKREQLKNGG
LGVVSDAI F
ELGKSLSAFNLDDTEVALLQAVLLMSTDRSGLLCVDKI EKSQEAYLLAFEHYVNHRKH NIPHFWPKLLMKEREVQ
SSILYKGAAAEGRPGGSLGVHPEGQQLLGMHVVQGPQVRQLEQQLGEAGSLOGPVLQHQSPKSPQQRLLELL
H RSG I LHARAVCG E DDSS EADS PSSS EEEP EVC ED LAG NAASP
AlFM1 095831 Apoptosis-inducing factor 1, mitochondrial Nucleotide Sequence (Seq ID No. 14):

>P003305_0311_0311_tube_AIFM1_9131_Apoptosis-inducing factor, mitochondrion-associated, 1 [Homo sapiens]_NM_001130846.2 0 0 0 0 0 0 0 ATGGAAAAGGTCCG CCGCGAGGGTGTCAAGGTCATGCCCAACGCTATCGTG CAGTCCGTGGGCGTGTCCT
CCGGCAAGCTGCTGATCAAGCTGAAGGACGGTCGCAAGGTGGAAACCGACCACATCGTGGCTGCTGTGGG
CCTCGAGCCCAACGTCGAGCTGGCTAAGACCGGTGGCCTCGAGATCGACTCCGACTTCGGTGGTTTCCGT
GTGAACGCTGAGCTGCAGGCTCGTTCCAACATCTGGGTGGCCGGCGACGCTGCTTGCTTCTACGACATCAA
GCTGGGTCGTCGTCGTGTCGAGCACCACGACCACGCTGTGGTGTCCGGTCGTCTGGCTGGCGAGAACATG
ACCGGTGCTGCTAAGCCCTACTGGCACCAGTCCATGTTCTGGTCCGACCTGGGTCCCGACGTGGGTTACG
AGGCTATCGGCCTGGTGGACTCCTCCCTGCCCACCGTGGGAGTGTTCGCTAAGGCTACCGCTCAGGACAA
CCCCAAGTCCGCTACCGAGCAGTCCGGCACCGGTATCCGTTCCGAGTCCGAGACTGAGTCCGAGGCTTCC
GAGATCACCATCCCCCCCTCCACCCCCGCTGTGCCTCAAGCTCCTGTGCAGGGCGAGGACTACGGCAAGG
GTGTCATCTTCTACCTGCGTGACAAGGTGGTCGTGGGTATCGTGCTGTGGAACATCTTCAACCGTATGCCTA
TCGCCCGCAAGATCATCAAGGACGGCGAGCAGCACGAGGACCTGAACGAGGTGGCCAAGCTGTTCAACAT
CCACGAGGAC
Protein Sequence (Seq ID No. 35):
>sp10958311AIFM1_HUMAN Apoptosis-inducing factor 1, mitochondrial OS=Homo sapiens OX=9606 GN=AIFM1 PE=1 SV=1 M FRCGG LAAGALKQKLVP LVRTVCVRSP RQ RN RLPG NLFQRWHVP
LELQMTRQMASSGASGGKIDNSVLVLIV
G LSTVGAGAYAYKTMKEDEKRYN ERISGLG LTPEQKQKKAALSASEG EEVPQ DKAPSH VP F
LLIGGGTAAFAAA
RSIRARDPGARVLIVSEDPELPYMRPPLSKELWFSDDPNVTKTLRFKOWNGKERSIYFQPPSFYVSAQDLPHIEN
GGVAVLTGKKVVQLDVR DNMVKLN DGSQITYEKCLI ATGGTPRSLSAI DRAGAEVKSRTTLFRKI
GDFRSLEKI SR
EVKSITI I GGGFLGSELACALGRKARALGTEVIQ LFP EKG NMGKI
LPEYLSNWTMEKVRREGVKVMPNAIVQSVGV
SSGKLLI KLKDGRKVETDH IVAAVG LEP NVELAKTGG LEI DSDFG GFRVNAELOARSNIWVAG DAAC
FYDI KLG RR
RVEHHDHAVVSGRLAGENMTGAAKPYWHQSMFWSDLGPDVGYEAIGLVDSSLPTVGVFAKATAQDNPKSATE
QSGTG I RSESETESEASEITI PPSTPAVPQAPVQGEDYG KGVI FYLRDKVVVG IVLWN I FN RM P
IARKI I KDG EQH E
DLNEVAKLFNIHED
ODC1 P11926 0 rnithi ne decarboxylase Nucleotide Sequence (Seq ID No. 15):
>P000568_SIG_SIG1-3_0DC1_4953_Homo sapiens ornithine decarboxylase 1 BCO25296.1 AAH25296.1 P11926 62117 0 1386 0 1383 ATGAACAACTTTGGTAATGAAGAGTTTGACTGCCACTTCCTCGATGAAGGTTTTACTGCCAAG GACATTCTG
GACCAGAAAATTAATGAAGTTTCTTCTTCTGATGATAAGGATGCCTTCTATGTGGCAGACCTGGGAGACATT
CTAAAGAAACATCTGAGGTGGTTAAAAGCTCTCCCTCGTGTCACCCCCTTTTATGCAGTCAAATGTAATGATA
GCAAAGCCATCGTGAAGACCCTTGCTGCTACCGGGACAGGATTTGACTGTGCTAGCAAGACTGAAATACAG
TTGGTGCAGAGTCTGGGGGTGCCTCCAGAGAGGATTATCTATGCAAATCCTTGTAAACAAGTATCTCAAATT
AAGTATGCTGCTAATAATGGAGTCCAGATGATGACTTTTGATAGTGAAGTTGAGTTGATGAAAGTTGCCAGA
GCACATCCCAAAGCAAAGTTGGTTTTGCGGATTGCCACTGATGATTCCAAAGCAGTCTGTCGTCTCAGTGTG
AAATTCGGTGCCACGCTCAGAACCAGCAGGCTCCTTTTGGAACGGGCGAAAGAGCTAAATATCGATGTTGT
TGGTGTCAGCTTCCATGTAGGAAGCGGCTGTACCGATCCTGAGACCTTCGTGCAGGCAATCTCTGATGCCC
GCTGTGTTTTTGACATGGGGGCTGAGGTTGGTTTCAGCATGTATCTGCTTGATATTGGCGGTGGCTTTCCTG
GATCTGAGGATGTGAAACTTAAATTTGAAGAGATCACCGGCGTAATCAACCCAGCGTTGGACAAATACTTTC
CGTCAGACTCTGGAGTGAGAATCATAGCTGAGCCCGGCAGATACTATGTTGCATCAGCTTTCACGCTTGCA
GTTAATATCATTGCCAAGAAAATTGTATTAAAGGAACAGACGGGCTCTGATGACGAAGATGAGTCGAGTGAG
CAGACCTTTATGTATTATGTGAATGATGGCGTCTATGGATCATTTAATTGCATACTCTATGACCACGCACATG
TAAAGCCCCTTCTGCAAAAGAGACCTAAACCAGATGAGAAGTATTATTCATCCAGCATATGGGGACCAACAT
GTGATGGCCTCGATCGGATTGTTGAGCGCTGTGACCTGCCTGAAATGCATGTG GGTGATTGGATGCTCTTT
GAAAACATGGGCGCTTACACTGTTGCTGCTGCCTCTACGTTCAATGGCTTCCAGAGGCCGACGATCTACTAT
GTGATGTCAGGGCCTGCGTGGCAACTCATGCAGCAATTCCAGAACCCCGACTTCCCACCCGAAGTAGAGGA
ACAG GATGCCAGCACCCTGCCTGTGTCTTGTGCCTGGGAGAGTGGGATGAAACGCCACAGAGCAGCCTGT
GCTTCGGCTAGTATTAATGTG

Protein Sequence (Seq ID No. 36):
>spIP119261DCOR_HUMAN Ornithine decarboxylase OS=Homo sapiens OX=9606 GN=ODC1 PE=1 SV=2 MNNFGNEEFDCHFLDEGFTAKDI LDQKINEVSSSDDKDAFYVADLGDI LKKHLRWLKALPRVTPFYAVKCNDSKAI

VKTLAATGTGFDCASKTEIQLVQSLGVPPERIIYANPCKQVSQIKYAANNGVQMMTFDSEVELMKVARAHPKAKL
VLRIATDDSKAVCRLSVKFGATLRTSRLLLERAKELNIDVVGVSFHVGSGCTDPETFVQAISDARCVFDMGAEVG
FSMYLLDIGGGFPGSEDVKLKFEEITGVINPALDKYFPSDSGVRIIAEPGRYYVASAFTLAVNIIAKKIVLKEQTGSD

DEDESSEQTFMYYVNDGVYGSFNCI LYDHAHVKPLLQKRPKPDEKYYSSSIWGPTCDGLDRIVERCDLPEMHVG
DWMLFENMGAYTVAAASTFNGFORPTIYYVMSGPAWQLMQQFQNPDFPPEVEEQDASTLPVSCAWESGMKR
H RAACASASI NV
RPS6KA4 075676 Ribosomal protein S6 kinase alpha-4 Nucleotide Sequence (Seq ID No. 16):
>P001321 Q305 Q305p4 RPS6KA4 8986 Homo sapiens RPS6KA4 ribosomal protein S6 kinase, 90kDa, polypeptide 4_BC047896_AAH47896_075676_0_0_1575_0_1572 ATGGGGGACGAGGACGACGATGAGAGCTGCGCCGTGGAGCTGCGGATCACAGAAGCCAACCTGACCGGG
CACGAGGAGAAGGTGAGCGTGGAGAACTTCGAGCTGCTCAAGGTGCTGGGCACGGGAGCCTACGGCAAG
GTGTTCCTGGTGCGGAAGGCGGGCGGGCACGACGCGGGGAAGCTGTACGCCATGAAGGTGCTGCGCAAG
GCGGCGCTGGTGCAGCGCGCCAAGACGCAGGAGCACACGCGCACCGAGCGCTCGGTGCTGGAGCTGGT
GCGCCAGGCGCCCTTCCTGGTCACGCTGCACTACGCTTTCCAGACGGATGCCAAGCTGCACCTCATCCTG
GACTATGTGAGCGGCGGGGAGATGTTCACCCACCTCTACCAGCGCCAGTACTTCAAGGAGGCTGAGGTGC
GCGTGTATGGGGGTGAGATCGTGCTGGCCCTGGAACACCTGCACAAGCTCGGCATCATTTACCGAGACCT
GAAACTGGAGAATGTGCTGCTGGACTCCGAGGGCCACATTGTCCTCACGGACTTCGGGCTGAGCAAGGAG
TTCCTGACGGAGGAGAAAGAGCGGACCTTCTCCTTCTGTGGCACCATCGAGTACATGGCCCCCGAAATCAT
CCGTAGCAAGACGGGGCATGGCAAGGCTGTGGACTGGTGGAGCCTG GGCATCTTGCTCTTCGAGCTGCTG
ACGGGGGCCTCGCCCTTCACCCTGGAGGGCGAGAGGAACACGCAGGCTGAGGTGTCTCGACGGATCCTG
AAGTGCTCCCCTCCCTTCCCCCCTCGGATCGGGCCCGTGGCGCAG GACCTGCTGCAGCGGCTGCTTTGTA
AGGATCCTAAGAAGCGATTGGGCGCGGGGCCCCAGGGGGCACAAGAAGTCCGGAACCATCCCTTCTTCCA
GGGCCTCGATTGGGTGGCTCTGGCTGCCAGGAAGATTCCAGCCCCATTCCGGCCCCAAATCCGCTCAGAG
CTGGATGTGGGCAACTTTGCGGAGGAATTCACTCGGCTGGAGCCTGTCTACTCACCCCCTGGCAGCCCCC
CACCTGGGGACCCCCGAATCTTTCAGGGATACTCCTTTGTGGCACCCTCCATTCTCTTTGACCACAACAACG
CGGCCGAGATCATGTGCAAAATCCGCGAGGGGCGCTTCTCCCTTGACGGGGAGGCCTGGCAGGGTGTATC
CGAGGAAGCCAAGGAGCTGGTCCGAGGGCTCCTGACCGTGGACCCCGCCAAGCGGCTGAAGCTCGAGGG
ACTGCGGGGCAGCTCGTGGCTGCAGGACGGCAGCGCGCGCTCCTCGCCCCCGCTCCGGACGCCCGACGT
GCTCGAGTCCTCTGGGCCCGCAGTGCGCTCGGGTCTCAACGCCACCTTCATGGCATTCAACCGGGGCAAG
CGGGAGGGCTTCTTCCTGAAGAGCGTGGAGAACGCACCCCTGGCCAAGCGGCGGAAGCAGAAGCTGCGG
AGCGCCACCGCCTCCCGCCGGGGCTCCCCTGCACCAGCCAACCCGGGCCGAGCCCCCGTCGCCGCCAAA
GGGGCCCCCCGCCGAGCCAACGGCCCCCTGCCCCCCTCC
Protein Sequence (Seq ID No. 37):
>sp1075676IKS6A4 HUMAN Ribosomal protein S6 kinase alpha-4 OS=Homo sapiens OX=9606 GN=RPS6KA4 PE=1 SV=1 MGDEDDDESCAVELRITEAN LTG H EEKVSVEN FELLKVLGTGAYG KVFLVRKAGGH DAG KLYAM
KVLRKAALVQ
RAKTQEHTRTERSVLELVRQAPFLVTLHYAFQTDAKLHLI LDYVSGGEMFTHLYQRQYFKEAEVRVYGGEIVLAL
EH LHKLG IIYRDLKLENVLLDSEG HIVLTDFGLSKEFLTEEKERTFSFCGTIEYMAPEI IRSKTG
HGKAVDWWSLG I L
LFELLTGASPFTLEGERNTQAEVSRRILKCSPPFPPRIGPVAQDLLQRLLCKDPKKRLGAGPQGAQEVRNHPFFQ
GLDWVALAARKIPAPFRPQIRSELDVGNFAEEFTRLEPVYSPPGSPPPGDPRIFQGYSFVAPSILFDHNNAVMTD
GLEAPGAGDRPGRAAVARSAMMQDSPFFQQYELDLREPALGQGSFSVCRRCRQRQSGQEFAVKILSRRLEAN
TOREVAALRLCOSHPNVVNLHEVHHDQLHTYLVLELLRGGELLEHIRKKRHFSESEASOILRSLVSAVSFMHEEA
GVVHRDLKPENILYADDTPGAPVKIIDFGFARLRPOSPGVPMQTPCFTLQYAAPELLAQQGYDESCDLWSLGVIL
YMMLSGQVPFQGASGQGGQSQAAEIMCKIREGRFSLDGEAWQGVSEEAKELVRGLLTVDPAKRLKLEGLRGSS
WLQDGSARSSPPLRTPDVLESSGPAVRSGLNATFMAFNRGKREGFFLKSVENAPLAKRRKQKLRSATASRRGS
PAPANPGRAPVASKGAPRRANGPLPPS

EEF1 D P29692 Elongation factor 1-delta Nucleotide Sequence (Seq ID No. 17):
>P001467 CAG CAGp2 EEF1 D 1936 Homo sapiens Homo sapiens eukaryotic translation elongation factor 1 delta (guanine nucleotide exchang BC007847.2 AAH07847.1 P29692 0 0 1944 0 1941 ATGAGGAGCGGGAAGGCCTCCTGCACCCTGGAGACCGTGTGGGAAGACAAGCACAAGTATGAGGAGGCC
GAGCGGCGCTTCTACGAACACGAGGCCACACAGGCGGCCGCCTCCGCCCAGCAGCTGCCAGCCGAGGGG
CCAGCCATGAATGG GCCCGGCCAGGACGACCCTGAGGACGCTGATGAGGCGGAAGCCCCTGACGGCGGC
AGCAGGCGTGATCCCAGGAAGAGCCAGGACAGCAGGAAGCCCCTGCAGAAAAAGAGGAAGCGCTCCCCC
AAGAGCGGGCTCGGCCCCGCGGACCTGGCCCTCCTGGGCCTCTCGGCCGAACGTGTGTGGCTGGACAAG
TCACTTTTCGACCAGGCAGAGAGCTCCTACCGCCAGAAGCTGGCAGATGTGGCTGCCCAGGCAGCCTGGC
CTCCTGCCTTGGCCCCTTGGGGTCTCTGCACCCATGGAAACCAGGTGGCCTGCCACCACGTGACCTGGGG
GATCTGGGTCAACAAGTCCTCCTTCGACCAGGCTGAGCGGGCCTTCGTGGAGTGGTCTCAGGCCCTGTTG
CTGGCCCCCGAGGGCAGCCGCAGGCAGGGGACTCCCAACACAGGCCAGCAGGTGGCCGTCCCCGACCTG
GCCCACCAGCCCAGCCCACCGGTCAATGGCCAGCCCCCGCTGGGCAGCCTGCAGGCACTGGTTCGGGAG
GTGTGGCTGGAGAAGCCCCGGTATGATGCAGCCGAGAGGGGCTTCTACGAGGCCCTGTTTGACGGCCATC
CCCCAG G GAAGGTGCGCCTGCAAGAGCGAGCCGGCCTGGCCGAG G GTGCCC GGCG GGGCCGCAGAGAC
CGGCGGGGCCGCAACATCTTAGGGAACAAGCG GGCCGGGCTGCGACGGGCCGATGGGGAGGCCCCCTC
TGCCTTGCCCTACTGTTACTTCCTGCAGAAGGATGCAGAGGCCCCCTGGCTCAGCAAGCCTGCCTACGACA
GCGCCGAGTGCCGCCACCACGCTGCCGAGGCCCTGCGTGTGGCCTGGTGCCTCGAAGCTGCCTCCCTGT
CTCACCGACCCGGTCCTCGGTCTGGCCTGTCCGTGTCCAGCCTGAGACCCAACAGAAAAATGGCTACAAAC
TTCCTAGCACATGAGAAGATCTGGTTCGACAAGTTCAAATATGACGACGCAGAAAGGAGATTCTACGAGCAG
ATGAACGGGCCTGTGGCAGGTGCCTCCCGCCAGGAGAACGGCGCCAGCGTGATCCTCCGTGACATTGCGA
GAGCCAGAGAGAACATCCAGAAATCCCTGGCTGGAAGCTCAGGCCCCG GGGCCTCCAGCGGCACCAGCG
GAGACCACGGTGAGCTCGTCGTCCGGATTGCCAGTCTGGAAGTGGAGAACCAGAGTCTGCGTGGCGTGGT
ACAGGAGCTGCAGCAGGCCATCTCCAAGCTGGAGGCCCGGCTGAACGTGCTGGAGAAGAGCTCGCCTGG
CCACCGGGCCACGGCCCCACAGACCCAGCACGTATCTCCCATGCGCCAAGTGGAGCCCCCAGCCAAGAAG
CCAGCCACACCAGCAGAG GATGACGAG GATGATGACATTGACCTGTTTGGCAGTGACAATGAGGAGGAGG
ACAAG GAG GCGGCACAGCTGCG G GAG GAGCGGCTACGGCAGTACGCGGAGAAGAAGGCCAAGAAGCCTG
CACTGGTGGCCAAGTCCTCCATCCTGCTGGATGTCAAGCCTTG GGATGATGAGACGGACATGGCCCAGCT
GGAGGCCTGTGTGCGCTCTATCCAGCTGGACGGGCTGGTCTGGGGGGCTTCCAAGCTGGTGCCCGTGGG
CTACGGTATCCGGAAGCTACAGATTCAGTGTGTGGTGGAGGACGACAAGGTGGGGACAGACTTGCTGGAG
GAGGAGATCACCAAGTTTGAGGAGCACGTGCAGAGTGTCGATATCGCAGCTTTCAACAAGATC
Protein Sequence (Seq ID No. 38):
>spIP296921EF1D_HUMAN Elongation factor 1-delta OS=Homo sapiens OX=9606 GN=EEF1D PE=1 SV=5 MATNFLAHEKIWFDKFKYDDAERRFYEQMNGPVAGASRQENGASVILRDIARARENIQKSLAGSSGPGASSGTS
GDHGELVVRIASLEVENQSLRGVVQELQQAISKLEARLNVLEKSSPGHRATAPQTQHVSPMRCWEPPAKKPATP
AEDDEDDDIDLFGSDNEEEDKEAAQLREERLRQYAEKKAKKPALVAKSSILLDVKPWDDETDMAQLEACVRSIQL
DGLVWGASKLVPVGYGIRKLQIQCVVEDDKVGTDLLEEEITKFEEHVQSVDIAAFNKI
KLF10 013118 Krueppel-like factor 10 Nucleotide Sequence (Seq ID No. 18):
>P000598_TRN_TRNp1_TIEG_7071_Homo sapiens TGFB inducible early growth response_BC011538.1_AAH11538.1_0530U8_0_0_1410_0_1407 ATGGAGGAAAGAATGGAAATGATTTCTGAAAGGCCAAAAGAGAGTATGTATTCCTGGAACAAAACTGCAGAG
AAAAGTGATTTTGAAGCTGTAGAAGCACTTATGTCAATGAGCTGCAGTTGGAAGTCTGATTTTAAGAAATACG
TTGAAAACAGACCTGTTACACCAGTATCTGATTTGTCAGAGGAAGAGAATCTGCTTCCGGGAACACCTGATT
TTCATACAATCCCAGCATTTTGTTTGACTCCACCTTACAGTCCTTCTGACTTTGAACCCTCTCAAGTGTCAAAT
CTGATGGCACCAGCGCCATCTACTGTACACTTCAAGTCACTCTCAGATACTGCCAAACCTCACATTG CCGCA
CCTTTCAAAGAGGAAGAAAAGAGCCCAGTATCTGCCCCCAAACTCCCCAAAGCTCAGGCAACAAGTGTGAT
TCGTCATACAGCTGATGCCCAGCTATGTAACCACCAGACCTGCCCAATGAAAGCAGCCAGCATCCTCAACTA
TCAGAACAATTCTTTTAGAAGAAGAACCCACCTAAATGTTGAGGCTGCAAGAAAGAACATACCATGTGCCGC
TGTGTCACCAAACAGATCCAAATGTGAGAGAAACACAGTGGCAGATGTTGATGAGAAAGCAAGTGCTGCAC

TTTATGACTTTTCTGTGCCTTCCTCAGAGACGGTCATCTGCAGGTCTCAGCCAGCCCCTGTGTCCCCACAAC
AGAAGTCAGTGTTGGTCTCTCCACCTGCAGTATCTGCAGGGGGAGTGCCACCTATGCCGGTCATCTGCCAG
ATGGTTCCCCTTCCTGCCAACAACCCTGTTGTGACAACAGTCGTTCCCAGCACTCCTCCCAGCCAGCCACC
AGCCGTTTGCCCCCCTGTTGTGTTCATGGGCACACAAGTCCCCAAAGGCGCTGTCATGTTTGTGGTACCCC
AGCCCGTTGTGCAGAGTTCAAAGCCTCCGGTGGTGAGCCCGAATGGCACCAGACTCTCTCCCATTGCCCCT
GCTCCTGGGTTTTCCCCTTCAGCAGCAAAAGTCACTCCTCAGATTGATTCATCAAGGATAAGGAGTCACATC
TGTAGCCACCCAGGATGTGGCAAGACATACTTTAAAAGTTCCCATCTGAAGGCCCACACGAG GACGCACAC
AGGAGAAAAGCCTTTCAGCTGTAGCTGGAAAGGTTGTGAAAGGAGGTTTGCCCGTTCTGATGAACTGTCCA
GACACAGGCGAACCCACACGGGTGAGAAGAAATTTGCGTGCCCCATGTGTGACCGGCGGTTCATGAGGAG
TGACCATTTGACCAAGCATGCCCGGCGCCATCTATCAGCCAAGAAGCTACCAAACTGGCAGATGGAAGTGA
GCAAGCTAAATGACATTGCTCTACCTCCAACCCCTGCTCCCACACAG
Protein Sequence (Seq ID No. 39):
>splQ131181KLF10_HUMAN Krueppel-like factor 10 OS=Homo sapiens OX=9606 GN=KLF10 PE=1 SV=1 MLNFGASLQQTAEERMEMISERPKESMYSWNKTAEKSDFEAVEALMSMSCSWKSDFKKYVENRPVTPVSDLSE
EENLLPGTPDFHTI PAFCLTP PYS PSD FE PSQVSN LMAPAPSTVH FKSLSDTAKPH I AAP FKE E E
KS PVSAPKL P K
AQATSVIRHTADAQLCNHQTCPMKAASILNYQNNSFRRRTHLNVEAARKNIPCAAVSPNRSKCERNTVADVDEK
ASAALYD FSVPSS ETVIC RSOPAPVS POO KSVLVSP PAVSAGGVP PM PVI COMVP L PAN N
PVVTTVVPSTP PSQ
P PAVCP PVVFMGTQVPKGAVMFVVPQPVVOSSKP PVVSP NGTR LS P IAPAP G FSPSAAKVT PQ I
DSS R I RSH ICS
HPGCGKTYFKSSHLKAHTRTHTGEKPFSCSWKGCERRFARSDELSRHRRTHTGEKKFACPMCDRRFMRSDHL
TKHAR RH LSAKKLP NWQMEVSKLN DI AL PPTPAPTQ
EPHA2 P29317 Ephrin type-A receptor 2 Nucleotide Sequence (Seq ID No. 19)-> P003284_Q311_Q 311_tube_E P HA2_1969_0_NM_004431.3_0_P29317_0 ATGGAACTGCAGGCTGCTCGTGCTTGCTTCGCTCTGCTGTGGGGTTGCGCTTTGGCTGCTGCAGCTGCTGC
TCAGGGCAAAGAGGTGGTCCTGCTGGACTTCGCTGCTGCCGGTGGCGAACTGGGATGGCTGACTCACCCT
TACGGCAAGGGCTGGGACCTGATGCAGAACATCATGAACGACATGCCCATCTACATGTACTCCGTGTGCAA
CGTGATGTCCGGCGACCAGGACAACTGGCTGCGTACCAACTGGGTGTACCGTGGCGAGGCTGAGCGCATC
TTCATCGAGCTGAAGTTCACCGTGCGCGACTGCAACTCCTTCCCTGGTG GTGCTTCCAGCTGCAAAGAG AC
TTTCAACCTGTACTACG CTGAGTCCG ACCTG GACTAC G GCACCAACTTCCAG AAG CGTCTGTTCACCAAG
AT
CGACACTATCGCTCCCGACGAGATCACCGTGTCCTCCGACTTCGAGGCTCGTCACGTGAAGCTGAACGTCG
AGGAACGCTCCGTGGGTCCCCTGACCCGCAAGGGATTCTACCTGGCTTTCCAGGACATCGGTGCTTGCGT
GGCCCTGCTGTCCGTGCGTGTGTACTACAAGAAGTGCCCCGAGCTGCTCCAGGGCCTGGCTCACTTCCCT
GAGACTATCGCTGGTTCCGACGCTCCCTCCCTGGCTACCGTTGCTGGTACTTGCGTGGACCACGCTGTGGT
GCCACCTGGTGGCGAGGAACCTCGTATGCACTGCGCTGTGGACGGCGAGTGGCTGGTGCCTATCGGTCAA
TGCCTGTGCCAGGCTGGTTACGAGAAGGTCGAGGACGCTTGCCAGGCTTGCTCCCCCGGTTTCTTCAAGTT
CGAGGCTTCCGAGTCCCCCTGCCTGGAATGCCCTGAACACACCCTGCCTTCCCCAGAGGGTGCTACCTCCT
GCGAGTGCGAAGAGGGCTTCTTCCGTGCTCCCCAGGACCCCGCTTCTATGCCTTGCACCCGTCCTCCCTCC
GCTCCCCACTACCTGACTGCTGTCGGCATGGGTGCTAAGGTCGAGCTGCGTTGGACCCCCCCTCAGGATT
CTGGTGGTCGCGAGGACATCGTCTACTCCGTGACCTGCGAGCAGTGCTGGCCTGAGTCTGGCGAATGCGG
TCCCTGCGAGGCTTCTGTGCGCTACTCTGAGCCTCCTCACGGCCTGACCCGTACCTCTGTGACCGTGTCCG
ACCTCGAGCCCCACATGAACTACACCTTCACCGTCGAGGCCCGTAACGGTGTCTCCGGACTGGTCACCTCC
CGTTCCTTCCGTACCGCTTCCGTGTCCATCAACCAGACCGAGCCCCCCAAAGTGCGCCTGGAAGGACGTTC
TACCACCTCCCTGTCCGTGTCTTGGTCCATCCCCCCACCTCAGCAGTCCCGTGTGTGGAAGTACGAAGTGA
CCTACCGCAAGAAG GGCGACTCCAACTCTTACAACGTGCGTCGTACCGAGGGTTTCAGCGTGACCCTGGAC
GACCTGGCTCCCGACACCACCTACCTGGTGCAAGTGCAGGCTCTGACCCAAGAGGGCCAGGGTGCTGGTT
CCAAGGTGCACGAGTTCCAGACCCTGTCCCCCGAGGGTTCCGGAAACTTGGCTGTGATCGGCGGTGTCGC
TGTGGGTGTCGTGCTGCTGTTGGTGCTGGCTGGTGTCGGCTTCTTCATCCACCGTCGTCGCAAGAACCAGC
GTGCTCGTCAGTCCCCTGAGGACGTGTACTTCTCCAAGTCCGAGCAGCTGAAGCCCCTCAAGACCTACGTG
GACCCCCACACTTACGAGGACCCCAACCAGGCTGTGCTCAAGTTCACTACCGAGATCCACCCCTCCTGCGT
GACCCGTCAGAAAGTGATCGGTGCTGGCGAGTTCGGCGAGGTGTACAAGGGAATGCTCAAGACTTCCTCC
GGCAAGAAAGAGGTGCCCGTCGCTATCAAGACCCTGAAGGCTGGCTACACCGAGAAGCAGCGTGTGGACT
TCCTGGGAGAGGCTGGTATCATGGGCCAGTTCTCCCACCACAACATCATCCGTCTGGAAGGTGTCATCTCC
AAGTACAAGCCCATGATGATCATTACCGAGTACATGGAAAACGGCGCCCTGGACAAGTTCCTGCGCGAGAA

GGATGGCGAGTTCTCCGTGCTGCAGCTCGTGGGAATGCTGCGTGGTATCGCTGCTGGCATGAAGTACCTG
GCCAACATGAATTACGTGCACAGGGACCTGGCTGCTCGCAACATCCTGGTCAACTCCAACCTCGTGTGCAA
GGTGTCAGACTTCGGCCTGTCCCGCGTGCTCGAGGACGATCCTGAGGCTACCTACACCACCTCCGGTGGA
AAGATCCCCATCCGTTGGACCGCTCCCGAGGCTATCTCTTACCGCAAGTTCACCTCCGCTTCCGACGTGTG
GTCCTTCGGTATCGTGATGTGGGAAGTGATGACCTACGGCGAGCGCCCCTACTGGGAGCTGTCTAACCAC
GAAGTCATGAAGGCTATCAACGACGGTTTCCGTCTGCCCACCCCTATGGACTGCCCCTCCGCTATCTACCA
GCTGATGATGCAATGCTGGCAGCAAGAGCGTGCTAGGCGTCCCAAGTTCGCTGACATCGTGTCTATCCTCG
ACAAGCTGATCCGCGCTCCTGACTCCCTGAAAACCCTGGCTGACTTCGACCCCCGTGTGTCCATCCGCCTG
CCTTCTACCTCTGGCTCCGAGGGTGTCCCTTTCCGTACTGTGTCCGAGTGGCTCGAGTCCATCAAGATGCA
GCAGTACACCGAGCACTTCATGGCTGCTGGTTACACCGCTATCGAGAAGGTGGTGCAGATGACCAACGACG
ACATCAAGCGTATCGGCGTGCGTCTGCCCGGTCACCAGAAGAGGATCGCTTACTCCCTGCTGGGCCTGAA
GGACCAAGTGAACACCGTGGGTATCCCCATC
Protein Sequence (Seq ID No. 40):
>spIP29317IEPHA2_HUMAN Ephrin type-A receptor 2 OS=Homo sapiens OX=9606 GN=EPHA2 PE=1 SV=2 MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNV
MSG DQDNWLRTNWVYRGEAERI Fl ELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKI
DTIAP
DEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSL
ATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPE
HTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQ

RLEGRSTTSLSVSWSI PP PQQSRVWKYEVTYRKKG DSNSYNVRRTEG FSVTLDDLAPDTTYLVQVQALTQEGQ
GAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYV
DP HTYEDP NQAVLKFTTEI HPSCVTRQKVIGAGEFGEVYKGM
LKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEA
G IMGQFSH H NI I RLEGVISKYKPMMI ITEYMENGALDKFLREKDG EFSVLQLVGMLRGIAAG
MKYLANMNYVH RDL
AARN I LVNSNLVCKVSDFGLSRVLEDDP EATYTTSGG KI PI RWTAPEAISYRKFTSASDVWSFGI
VMWEVMTYGE
RPYW ELSN H EVM KAI NDGFRLPTPMDCPSAIYQLMMQCWQQERARRPKFADIVSI LDKLI RAP DSLKT
LADFDP R
VSIRLPSTSGSEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDI KRIGVRLPG HQKRIAYSL LG
LK
DQVNTVGIPI
PRKAR1A P10644 cAMP-dependent protein kinase type l-alpha regulatory subunit Nucleotide Sequence (Seq ID No. 20):
>P000113_CAN_CAN1-1_PRKAR1A_5573_Homo sapiens protein kinase cAMP-dependent regulatory type I
alpha (tissue specific e BC036285.1 AAH36285.1 P10644 0 0 1146 0 1143 ATGGAGTCTGGCAGTACCGCCGCCAGTGAGGAGGCACGCAGCCTTCGAGAATGTGAGCTCTACGTCCAGA
AGCATAACATTCAAGCGCTGCTCAAAGATTCTATTGTGCAGTTGTGCACTGCTCGACCTGAGAGACCCATGG
CATTCCTCAGGGAATACTTTGAGAGGTTGGAGAAGGAGGAGGCAAAACAGATTCAGAATCTGCAGAAAGCA
GGCACTCGTACAGACTCAAGGGAGGATGAGATTTCTCCTCCTCCACCCAACCCAGTGGTTAAAGGTAGGAG
GCGACGAGGTGCTATCAGCGCTGAGGTCTACACGGAGGAAGATGCGGCATCCTATGTTAGAAAGGTTATAC
CAAAAGATTACAAGACAATGGCCGCTTTAGCCAAAGCCATTGAAAAGAATGTGCTGTTTTCACATCTTGATGA
TAATGAGAGAAGTGATATTTTTGATGCCATGTTTTCGGTCTCCTTTATCGCAGGAGAGACTGTGATTCAGCAA
GGTGATGAAGGGGATAACTTCTATGTGATTGATCAAGGAGAGACGGATGTCTATGTTAACAATGAATGGGCA
ACCAGTGTTGGGGAAGGAGGGAGCTTTGGAGAACTTGCTTTGATTTATGGAACACCGAGAGCAGCCACTGT
CAAAGCAAAGACAAATGTGAAATTGTGGGGCATCGACCGAGACAGCTATAGAAGAATCCTCATGGGAAGCA
CACTGAGAAAGCGGAAGATGTATGAGGAATTCCTTAGTAAAGTCTCTATTTTAGAGTCTCTGGACAAGTGGG
AACGTCTTACGGTAGCTGATGCATTGGAACCAGTGCAGTTTGAAGATGGGCAGAAGATTGTGGTGCAGGGA
GAACCAGGGGATGAGTTCTTCATTATTTTAGAGGGGTCAGCTGCTGTGCTACAACGTCGGTCAGAAAATGAA
GAGTTTGTTGAAGTGGGAAGATTGGGGCCTTCTGATTATTTTGGTGAAATTGCACTACTGATGAATCGTCCT
CGTGCTGCCACAGTTGTTGCTCGTGGCCCCTTGAAGTGCGTTAAGCTGGACCGACCTAGATTTGAACGTGT
TCTTGGCCCATGCTCAGACATCCTCAAACGAAACATCCAGCAGTACAACAGTTTTGTGTCACTGTCTGTC
Protein Sequence (Seq ID No. 41):
>spIP106441KAPO_HUMAN cAMP-dependent protein kinase type I-alpha regulatory subunit OS=Homo sapiens OX=9606 GN=PRKAR1A PE=1 SV=1 MESGSTAASEEARSLRECELYVOKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKQIQNLQKAGTRT
DSREDEISPPPPNPVVKGRRRRGAISAEVYTEEDAASYVRKVIPKDYKTMAALAKAI EKNVLFSHLDDNERSDI FD

AMFSVSFIAGETVIQQG DEG D NFYVI DOGETDVYVN N EWATSVG EGGSFG
ELALIYGTPRAATVKAKTNVKLWGI
DRDSYRRILMGSTLRKRKMYEEFLSKVSI LESLDKWERLTVADALEPVQFEDGQKIVVQG EPG DEFFI I
LEGSAAV
LORRSENEEFVEVGRLGPSDYFGEIALLMNRPRAATVVARGPLKCVKLDRPRFERVLGPCSDILKRNIQQYNSFV
SLSV
EAPP 056P03 E2F-associated phosphoprotein Nucleotide Sequence (Seq ID No. 21):
>10001616_0106_0106p2_EAPP_55837_Homo sapiens chromosome 14 open reading frame 11_BC001245.1_AAH01245.1 xx 0 0 858 0 855 ATGAACCGGCTTCCGGATGACTACGACCCCTACGCGGTTGAAGAGCCTAGCGACGAGGAGCCGGCTTTGA
GCAGCTCTGAGGATGAAGTGGATGTGCTTTTACATGGAACTCCTGACCAAAAACGAAAACTCATCAGAGAAT
GTCTTACCGGAGAAAGTGAATCATCTAGTGAAGATGAATTTGAAAAGGAGATGGAAGCTGAATTAAATTCTA
CCATGAAAACAATGGAGGACAAGTTATCCTCTCTGGGAACTGGATCTTCCTCAGGAAATGGAAAAGTTGCAA
CAGCTCCGACAAGGTACTACGATGATATATATTTTGATTCTGATTCCGAGGATGAAGACAGAGCAGTACAGG
TGACCAAGAAAAAAAAGAAGAAACAACACAAGATTCCAACAAATGACGAATTACTGTATGATCCTGAAAAAGA
TAACAGAGATCAGGCCTGGGTTGATGCACAGAGAAGGGGTTACCATGGTTTGGGACCACAGAGATCACGTG
AACAACAGCCTGTTCCAAATAGTGATGCTGTCTTGAATTGTCCTGCCTGCATGACCACACTTTGCCTTGATTG
CCAAAGGCATGAATCATACAAAACTCAATATAGAGCAATGTTTGTAATGAATTGTTCTATTAACAAAGAGGAG
GTTCTAAGATATAAAGCCTCAGAGAACAG GAAGAAAAG G CG G GTCCATAAGAAGATGAG GTCTAACCAGGA
AGATGCTGCTGAGAAGGCAGAGACAGATGTGGAAGAAATCTATCACCCAGTCATGTGCACTGAATGTTCCA
CTGAAGTGGCAGTCTACGACAAGGATGAAGTCTTTCATTTTTTCAATGTTTTAGCAAGCCATTCC
Protein Sequence (Seq ID No. 42):
>splQ56P031EAPP_HUMAN E2F-associated phosphoprotein OS=Homo sapiens OX=9606 GN=EAPP PE=1 SV=4 MNRLPDDYDPYAVEEPSDEEPALSSSEDEVDVLLHGTPDOKRKLI RECLTGESESSSEDEFEKEMEAELNSTMK
TMEDKLSSLGTGSSSGNGKVATAPTRYYDDIYFDSDSEDEDRAVQVTKKKKKKOHKI PTNDELLYDPEKDNRDQ
AWVDAQRRGYHGLGPQRSRQQQPVPNSDAVLNCPACMTTLCLDCORHESYKTQYRAMFVMNCSINKEEVLRY
KASENRKKRRVHKKMRSNREDAAEKAETDVEEIYHPVMCTECSTEVAVYDKDEVFHFFNVLASHS

Claims (19)

Claims
1. A method for predicting a response to adalimumab from a sample extracted from a rheumatoid arthritis patient prior to treating the patient with adalimumab, said response being classified as a good response corresponding to anti-drug antibody negative or a poor response corresponding to anti-drug antibody positive, comprising the steps of:
(i) testing the sample for the presence of autoantibody biomarkers; and (ii) determining whether the patient will develop a good response or a bad response to treatment with adalimumab, based on the detection of said autoantibody biomarkers;
characterised in that said autoantibody biomarkers comprise autoantibodies to antigens SSB, TROVE2 and ZHX2, wherein ZHX2 is associated with the good response, and SSB and TROVE2 are associated with the poor response.
2. The method according to claim 1 wherein the autoantibody biomarkers further comprise autoantibodies to one or more antigens from the group comprising of PPARD, SPANXN2, HNRNPA2B, TRIB2, CEP55, SH3GL1, FN3K, PANK3, HPCAL1, THRA, AIFM1, ODC1, RPS6KA4, EEF1D, KLF10, EPHA2, PRKAR1A and EAPP.
3. The method according to claim 1 or 2 wherein the antigens are biotinylated proteins.
4. The method according to claim 3 wherein each biotinylated protein is formed from a Biotin Carboxyl Carrier Protein folding marker which is fused in-frame with a protein.
5. The method according to claim 3 or 4 wherein the biotinylated proteins are bound to a streptavidin-coated substrate.
6. The method according to claim 5 wherein the substrate comprises a hydrogel-forming polymer base layer.
7. The method according to any preceding claim wherein the antigens are exposed to a sample extracted from a person, such that autoantibody biomarkers from the sample may bind to the antigens.
8. The method according to claim 7 wherein the antigens are subsequently exposed to a fluorescently-tagged secondary antibody to allow the amount of any autoantibodies from the sample bound to the antigens to be determined.
9. The method according to claim 8 wherein the patient's response to treatment with adalimumab corresponds to the relative or absolute amount of autoantibodies from the sample specifically binding to the antigens.
10. The method according to any preceding claim wherein the sample comprises any or any combination of exosomes, blood, serum, plasma, urine, saliva, amniotic fluid, cerebrospinal fluid, breast milk, semen or bile.
11. The method according to any preceding claim wherein the steps are performed in vitro .
12. The method according to any preceding claim wherein the method comprises detecting upregulation/downregulation of one or more biomarkers.
13. A method for manufacturing a kit for predicting a response to adalimumab from a sample extracted from a rheumatoid arthritis patient prior to treating the patient with adal i mum ab, com pri sing the steps of:
for each antigen in a panel, cloning a biotin carboxyl carrier protein folding marker in-frame with a gene encoding the antigen and expressing the resulting biotinylated antigen;
binding the biotinylated antigens to addressable locations on one or more streptavidin-coated substrates, thereby forming an antigen array;
such that the amount of autoantibodies from the sample binding to the antigens on the panel can be determined by exposing the substrate to the sample and measuring the response;
characterised in that the antigens comprise SSB, TROVE2 and ZHX2.
14. The method according to claim 13 wherein the antigens further comprise one or more of PPARD, SPANXN2, HNRNPA2B, TRIB2, CEP55, SH3GL1, FN3K, PANK3, HPCAL1, THRA, AIFM1, ODC1, RPS6KA4, EEF1D, KLF10, EPHA2, PRKAR1A and EAPP.
15. A composition comprising a panel of antigens for predicting an immunogenic and/or therapeutic response to adalimumab in a rheumatoid arthritis patient who has not previously been treated with adalimumab, characterised in that the antigens comprise SSB, TROVE2 and ZHX2.
16. A composition according to claim 15 wherein the antigens further comprise one or more of PPARD, SPANXN2, HNRNPA2B, TRIB2, CEP55, SH3GL1, FN3K, PANK3, HPCAL1, THRA, AIFM1, ODC1, RPS6KA4, EEF1D, KLF10, EPHA2, PRKAR1A and EAPP.
17. A composition according to claim 15 or 16 wherein the antigens are biotinylated proteins.
18. A composition according to any of claims 1 5-1 7 wherein the amount of one or more autoantibody biomarkers binding in vitro to the antigens in a sample from a patient can be measured to predict the response.
19. A composition comprising a panel of autoantibody biomarkers for predicting an immunogenic and/or therapeutic response to adalimumab in a rheumatoid arthritis patient who has not previously been treated with adalimumab, wherein the level of the autoantibody biomarkers are measured in a sample collected from the patient;
characterised in that the autoantibody biomarkers are specific to antigens comprising SSB, TROVE2 and ZHX2.
CA3199484A 2020-11-18 2021-11-11 Biomarkers for predicting immunogenicity and therapeutic responses to adalimumab in rheumatoid arthritis patients Pending CA3199484A1 (en)

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